Adenovirus p53 compositions and methods

ABSTRACT

Disclosed are methods and compositions for the selective manipulation of gene expression through the preparation of retroviral expression vectors for expressing antisense sequences, such as K-ras oncogene antisense sequences, or sequences encoding a desired product, such as wild type p53 sequences. Preferred retroviral vectors of the present invention incorporate the β-actin promoter in a reverse orientation with respect to retroviral transcription. Preferred antisense RNA constructs of the present invention employ the use of antisense intron DNA corresponding to distinct intron regions of the gene whose expression is targeted for down-regulation. In an exemplary embodiment, a human lung cancer cell line (NCI-H460a) with a homozygous spontaneous K-ras mutation was transfected with a recombinant plasmid that synthesizes a genomic segment of K-ras in antisense orientation. Translation of the mutated K-ras mRNA was specifically inhibited, whereas expression of H-ras and N-ras was unchanged. A three-fold growth inhibition occurred in H460a cells when expression of the mutated ras p21 protein was down-regulated by antisense RNA and cells remained viable. The growth of H460a tumors in nu/nu mice was substantially reduced by expressed K-ras antisense RNA.

[0001] The present application is a continuation-in-part of U.S. Ser.No. 07/665,538, filed Mar. 6, 1991.

[0002] The government may own certain rights in the present inventionpursuant to NIH grants RO1 CA 45187 and CA 16672.

BACKGROUND OF THE INVENTION

[0003] 1. Field of the Invention

[0004] The present invention relates to methods and nucleic acid vectorcompositions for modifying gene expressing, involving the preparationand use of improved retroviral vectors which encode antisense RNAmolecules or, alternatively, transcriptionally active RNAs that encodeselected proteins. The retroviral constructs of the present inventionmay be employed for introducing desired gene expression units intoselected target cells, such as into tumor cells within individualsafflicted with cancer.

[0005] 2. Description of the Related Art

[0006] It is now well established that a variety of diseases, rangingfrom certain cancers to various genetic defects, are caused, at least inpart, by genetic abnormalities that result in either the over expressionof one or more genes, or the expression of an abnormal or mutant gene orgenes. For example, many forms of cancer in man are now known to be theresult of, at least indirectly, the expression of “oncogenes”. Oncogenesare genetically altered genes whose altered expression product somehowdisrupts normal cellular function or control (Spandidos, et al., 1989).

[0007] Most oncogenes studied to date have been found to be “activated”as the result of a mutation, often a point mutation, in the codingregion of a normal cellular gene or of a “protooncogene”, that resultsin amino acid substitutions in the protein expression product. Thisaltered expression product, in turn, exhibits an abnormal biologicalfunction that somehow takes part in the neoplastic process (Travali, etal., 1990). The underlying mutations can arise by various means, such asby chemical mutagenesis or ionizing radiation.

[0008] A number of oncogenes have now been identified and characterizedto varying degrees, including ras, myc, neu, raf, erb, src, fms, jun andabl (Travali, et al., 1990; Minna, 1989; Bishop, 1987). It is likelythat as our knowledge of tumori-genesis increases, additional oncogeneswill be identified and characterized. Many of the foregoing, includingras, myc and erbB, comprise families of genes, whose expression productbear sequence similarities to other members of the family (Shih, et al.,1984; Bos, 1989; Schwab, et al., 1985). In the case of many of thesegene families, it is typical that oncogenesis involves an activation ofonly one member of the family, with other “unactivated” members servinga role in normal cellular functions (Id.).

[0009] The study of DNA-mediated gene transfer has revealed theexistence of activated cellular oncogenes in a variety of human tumors(for review, see Cooper, et al., 1982). Oncogenes have been identifiedin human bladder, colon, lung and mammary carcinoma cell lines(Krontiris, et al., 1981; Murray, et al., 1981; Perucho, et al., 1981),promyelocytic leukemia (Murray, et al., 1981), neuroblastoma (Shimizu,et al., 1983) and sarcoma cell lines (Pulciani, et al., 1982), andvarious solid tumors including carcinomas of the lung, and pancreas(Pulciani, et al., 1982). Studies have demonstrated that varioustransforming genes detected by transfection correspond to activatedcellular homologues of retroviral oncogenes (Pulciani, et al., 1982;Der, et al., 1982; Parada, et al., 1982; Santos, et al., 1982), althoughothers have no known retroviral cognate (Tulciani, et al., 1982; Lane,et al., 1982).

[0010] The ras oncogene family has been perhaps the best characterizedto date (Barbacid, 1987; Bos, 1989). Most of the identified transforminggenes in human carcinomas have been a member of the ras gene family,which encode immunologically related proteins having a molecular weightof 21,000 (p21) (Ellis, et al., 1981; Papageorge, et al., 1982). Thisfamily is comprised of at least 3 members, one transduces as H-ras inthe Harvey strain of murine sarcoma virus (Ellis, et al., 1981), one asK-ras and Kirsten murine sarcoma virus (Ellis, et al., 1981), and oneidentified by low stringency hybridization to H-ras, termed N-ras(Shimizu, et al., 1983). As noted, all members of the ras gene familyencode closely related proteins of approximately 21,000 Daltons whichhave been designated p21s (Ellis, et al, 1981). The level of p21expression is similar in many different human tumor cells, independentof whether the cell contains an activated ras gene detectable bytransfection.

[0011] Nucleotide sequence analysis of the H-ras transforming gene ofthe EJ human bladder carcinoma has indicated that the transformingactivity of this gene is a consequence of a point mutation alteringamino acid 12 of p21 from glycine to valine (Tabin, et al., 1982).Studies of proteins encoded by K-ras genes activated in four human lungand colon carcinoma cell lines indicated that the transforming activityof K-ras in these human tumors was also a consequence of structuralmutations (Der and Cooper, 1983). Other mutations have been found toresult in ras gene activation as well. For example, the H-ras geneactivated in a lung carcinoma cell line encodes the normal amino acidposition 12 but is mutated at codon 61 to encode leucine rather thanglutamine (Yuasa, et al., 1983). An N-ras gene activated in a humanneuroblastoma cell line is also mutated at codon 61 but encodes lysinerather that glutamine (Taparowski, et al., 1983). Thus, studies such asthese have indicated that ras genes in human neoplasms are commonlyactivated by structural mutations, often point mutations, that thus faroccur at codon 12 or 61 with different amino acid substitutionsresulting in ras gene activation in different tumors.

[0012] Antisense RNA technology has been developed as one approach toinhibiting gene expression, particularly oncogene expression. An“antisense” RNA molecule is one which contains the complement of, andcan therefore hybridize with, protein-encoding RNAs of the cell. It isbelieved that the hybridization of antisense RNA to its cellular RNAcomplement can prevent expression of the cellular RNA, perhaps bylimiting its translatability. While various studies have involved theprocessing of RNA or direct introduction of antisense RNAoligonucleotides to cells for the inhibition of gene expression (Brown,et al., 1989; Wickstrom, et al., 1988; Smith, et al., 1986; Buvoli, etal., 1987), the more common means of cellular introduction of antisenseRNAs has been through the construction of recombinant vectors which willexpress antisense RNA once the vector is introduced into the cell.

[0013] A principal application of antisense RNA technology has been inconnection with attempts to affect the expression of specific genes. Forexample, Delauney, et al. have reported the use antisense transcripts toinhibit gene expression in transgenic plants (Delauney, et al., 1988).These authors report the down-regulation of chloramphenicol acetyltransferase activity in tobacco plants transformed with CAT sequencesthrough the application of antisense technology.

[0014] Antisense technology has also been applied in attempts to inhibitthe expression of various oncogenes. For example, Kasid, et al., 1989,report the preparation of recombinant vector construct employing Craf-1cDNA fragments in an antisense orientation, brought under the control ofan adenovirus 2 late promoter. These authors report that theintroduction of this recombinant construct into a human squamouscarcinoma resulted in a greatly reduced tumorigenic potential relativeto cells transfected with control sense transfectants. Similarly,Prochownik, et al., 1988, have reported the use of Cmyc antisenseconstructs to accelerate differentiation and inhibit G₁ progression inFriend Murine Erythroleukemia cells In contrast, Khokha, et al., 1989,discloses the use of antisense RNAs to confer oncogenicity on 3T3 cells,through the use of antisense RNA to reduce murine tissue inhibitor ormetalloproteinases levels.

[0015] Unfortunately, the use of current antisense technology oftenresults in failure, particularly where one seeks to selectively inhibita member of a gene family. One reason for this failure can be traced tothe high expression levels of antisense message that are apparentlyrequired for inhibition. Unfortunately, the requisite expression levelsof antisense message has not been generally achievable with existingconstructs. Problems have also arisen due to the similarity inunderlying DNA sequences, which results in the cross-hybridization ofantisense RNA, retarding the expression of genes required for normalcellular functions. An example is presented by Debus, et al., 1990, whoreported that in the case of ras oncogenes, antisense rasoligonucleotides kill both normal and cancer cells, which, of course, isnot a desired effect.

[0016] Another important “oncogene” is the gene encoding the p53cellular protein. The p53 gene is one of the most common targets forgenetic abnormalities in human tumors (Hollstein et al., 1991). Forexample, it has been reported that p53 mutations occur in allhistological types of lung cancer at frequencies of about 75% in smallcell lung cancer (SCLC) and about 50% in non small cell lung cancer(NSCLC) (Takahashi et al., 1991). Evidence suggests that p53 acts as a“tumor suppressor” gene, and its inactivation through mutation can leadto oncogenic development. In fact, a predominance of G to Ttransversions in p53 and ras mutations in lung cancer, as well asepidemiological data, supports a close association between smoking andp53 mutations in NSCLC have suggested that p53 is a candidate formolecular targets of genetic damage caused by cigarette smoke(Zakut-Houri et al., 1985).

[0017] One approach that has been suggested as a means of treatment ofsuch tumors is the introduction of so-called “wild-type” or non-mutatedp53 (wt-p53) into affected cells, e.g., through the use of retroviralvectors which encode the wild type protein (Takahashi et al., 1992; Leeet al., EP appl. publ. 0 475 623 A1). The vectors proposed by theseindividuals included a wt-p53 genes wherein the direction oftranscription of the encoded wt-p53 was in the same orientation as thatof the retroviral long terminal repeats (LTRs). Unfortunately, instudies conducted by the present inventors reported hereinbelow, theability of retroviral wt-p53 constructs prepared having such anorientation to suppress tumor growth was found to be less than optimal.Presumably, this shortcoming is the result of poor expression of thewt-p53 gene in the target cells.

[0018] Therefore, while it is clear that current gene therapy technologyshows potential promise as a means of external control of geneexpression, it is equally clear that it does suffer particular drawbacks, such as the need for high level expression and a lack ofselectivity where gene families are concerned. There is a particularneed, therefore, for a general approach to the design of gene therapyprotocols that will allow selective inhibition of gene expression, evenin the case of closely related genes.

SUMMARY OF THE INVENTION

[0019] The present invention, in a general and overall sense, addressesone or more of the foregoing or other shortcomings in the prior art byproviding a novel approach to the design of retroviral vectors for theintracellular delivery of selected genetic constructs in a manner whichallows their use to inhibit the expression of specific genes, or toreplace defective genes, in target cells.

[0020] The inventors believe that the approach offered by the presentinvention offers more specificity and selectivity than previousapproaches. Additionally, it is proposed that the present invention willallow that the development of vector technology for gene therapy havinga much improved ability to inhibit or provide for specific geneexpression, particularly in those instances where-one desires toselectively inhibit a particular gene over that of closely related genesor other members of a gene family, or where one desires to provide forthe expression of a specific gene.

[0021] A particularly surprising aspect of the invention, discussed inmore detail below, is the finding that by aligning the selectedpromoter/gene construct within the vector in an orientation that isreversed with respect to the direction of transcription of otherpromoters within the vector, one can achieve a dramatic increase intranscription of the introduced gene. Thus, where retroviral vectors areemployed, the promoter/gene construct should be aligned so as to effecttranscription in a direction that is opposite that of usual viraltranscription. In the case of retroviruses, a reverse orientation is onethat is opposite that of long terminal repeat transcription. While thisaffect was observed using the β-actin promoter and a retroviralexpression vector, the inventors believe that this phenomenon will beapplicable to other promoter/vector constructs for application in genetherapy.

[0022] In one specific embodiment, the invention concerns vectorconstructs for introducing wild type p53 genes (wt-p53) into affectedtarget cells suspected of having mutant p53 genes. These embodimentsinvolve the preparation of a gene expression unit wherein the wt-p53gene is placed under the control of the β-actin promoter, and the unitis positioned in a reverse orientation into a retroviral vector.

[0023] While aspects of the invention are exemplified through the use ofwt-p53 constructs, and their use in cancer treatment, it is proposedthat the invention is generally applicable to any situation where onedesires to achieve high level expression of a recombinant protein in atarget or host cell through the use of a retroviral expression vector.This could, for example, involve the use of a gene encoding arecombinant protein that confers a particular trait, such as the use ofwt-p53 to “replace” a trait that has been lost due to mutation, or couldbe used to introduce protein-encoding genes that one desires to use forpreparing proteins for other purposes, such as in recombinant proteinproduction procedures. While the nature of the gene introduced is notcritical to broader aspects of the invention, it should be mentionedthat in the context of cancer treatment modalities, a particular examplein addition to p53 replacement that is contemplated by the inventors isthe introduction of the retinoblastoma gene (rb).

[0024] In embodiments where inhibition or suppression of gene expressionis desired, antisense molecules will be employed. By preparing aconstruct that encodes an RNA molecule that is in antisense or“complementary” configuration with respect to the RNA readouts of thetarget gene, the construct will act to inhibit or suppress the ultimateexpression of the target gene, presumably by binding to the target RNAand thereby preventing its translation. In that the novel aspects ofthis part of the invention concerns the discovery of an improvedretroviral promoter construct, the invention is generally applicable toany antisense construct.

[0025] For certain applications in the context of antisense constructs,therefore, the antisense RNA that is produced will be complementary to aselected cellular gene, such as an oncogene sequence or some othersequence whose expression one seeks to diminish through antisenseapplication. While all or part of the coding sequence may be employed inthe context of antisense construction, the inventors have found thatparticular advantage may be taken where one employs in the antisenseconstruct an intron-complementary region that will bind to transcribedintrons contained in the targeted RNA. It has been found that the use ofintron-complementary regions not only improves the inherent inhibitorycharacteristics of the antisense molecule, but it also provides one theability to selectively inhibit one member of a gene family over another.This is due to the fact that while exon regions of members of genefamilies will often be similar, it is typically the case that the intronregions of these genes will be different.

[0026] Thus, in preferred aspects of the invention, antisense moleculeswill include a region that is complementary to and is capable ofhybridizing with an intron region of the gene whose expression is to beinhibited. The inclusion of intron-complementary regions in theantisense RNA constructs of the present invention is believed to be thekey to both an improved inhibitory capability as well as selectivity. Byway of theory, it is proposed that the use of antisense intron regionsprovides an improved capability for at least two reasons. It is knownthat the structure of intron RNA plays a role in RNA processing.

[0027] The inventors propose that antisense introns bind to “sense”intron regions found on the initial RNA transcript of the gene, andaffects proper RNA processing. Thus, subsequent translation ofprotein-coding RNAs into their corresponding proteins is retarded orprevented. The use of antisense introns are believed to provideselectivity of inhibition because the exon or “amino acid encoding”region of RNAs coding for closely related proteins are often themselvesclosely related. This may not be the case for the introns of closelyrelated genes. Thus, where intron regions between two genes aredistinct, antisense introns can be designed which will hybridizeselectively to a selected gene family member, and not to other familymembers, and thereby inhibit selectivity.

[0028] As used herein, the term “intron” is intended to refer to generegions that are transcribed into RNA molecules, but processed out ofthe RNA before the RNA is translated into a protein. In contrast, “exon”regions of genes are those regions which are transcribed into RNA andsubsequently translated into proteins.

[0029] Thus, where one seeks to selectively inhibit a particular gene orgenes over a related gene or genes, the inventors propose thepreparation and use of antisense RNA molecules which encode an intronregion or regions of the gene which one desires to inhibit selectively,that is distinct from intron regions of genes which one desires to leaveunaffected. A “distinct” intron region, as used herein, is intended torefer to an intron region that is sufficiently different from an intronregion of another gene such that no cross hybridization would occurunder physiologic conditions. Typically, where one intron exhibits asequence homology of no more than 20% with respect to a second intron,one would not expect hybridization to occur between antisense and senseintrons under physiologic conditions.

[0030] While it is generally preferred that antisense introns beprepared to be complementary to an entire intron of the gene to beinhibited, it is believed that shorter regions of complementarity can beemployed, so long as the antisense construct can be shown in vitro toinhibit expression of the targeted expression product. The inventorsbelieve that the most important intron regions in terms of thepreparation of antisense introns will be those regions closest tointron/exon junctions. This is the region where RNA processing takesplace. Thus, it is proposed that one will desire to include it in theantisense intron sufficient complementarity with regions within 50-100nucleotides of the intron/exon junction.

[0031] The inventors have found that some antisense exon sequences ofthe targeted gene can also be included in the antisense constructs ofthe present invention, so long as the resultant construct maintains itsselectivity, and will not seriously inhibit genes whose continuedfunction is relied upon by the cell for normal cellular activities. Theamount of antisense exon sequence included within the antisenseconstruct which can be tolerated will likely vary, depending on theparticular application envisioned. For example, antisense constructs fordown-regulation of K-ras expression have been prepared which includesequences complementary to exons II and III and all of intron II of theK-ras gene. These constructs contain antisense sequences to intron II ofK-ras, and selectively inhibit K-ras expression relative to H-ras orN-ras. Thus, in this instance, the inclusion of sequences complementaryto exons II and III of K-ras apparently did not result in thesignificant inhibition of the H-ras or N-ras genes, even though a 300nucleotide region of complementarity existed with exons of theunaffected genes.

[0032] One can readily test whether too much antisense exon DNA has beenincluded in antisense intron constructs of the present invention bysimply testing the constructs in vitro to determine whether normalcellular function is affected or whether the expression of related geneshaving complementary sequences are affected.

[0033] In connection with these aspects of the invention, it is proposedthat the antisense constructs of the present invention, whether they bethe antisense RNA molecules (i.e., oligonucleotides) or nucleic acidmolecules which encode for antisense RNA molecules, will have theirprincipal application in connection with the down-regulation of oncogeneexpression.

[0034] The most preferred oncogenes for application of the presentinvention will be those which exist as a family of genes, where onedesires to selectively inhibit one member of a family over othermembers. In this regard, one may mention by way of example, the ras,myc, erb or jun families of oncogenes. Certain of these, such as the rasfamily, involves the activation of protooncogenes by a point mutation,which apparently results in the expression of a biologically abnormalproduct.

[0035] In aspects that relate to the use of intron sequences, thepresent invention contemplates that antisense intron RNA can either beapplied directly to cells, in the form of oligonucleotides incorporatingantisense intron sequences, or by introducing into the cell nucleic acidsequences that will encode the desired antisense construct in the formof retroviral constructs. In the former case, it has been shown byothers that antisense oligonucleotides can successfully traversecellular membranes. The present inventors envision that such an approachmay be an option to therapy, particularly where the antisenseoligonucleotides are successfully packaged to maintain their stabilityin circulation, for example, by liposome encapsulation.

[0036] Other techniques for direct insertion in the cells include, byway of example, electroporation, or calcium phosphate transfection.Furthermore, where one desires to treat conditions of the bone marrow,bone marrow cells can be successfully removed from the body, treatedwith antisense constructs, and replaced into the body similar to theadoptive immunotherapy approach employed in IL-2 treatment.

[0037] In broader aspects of the invention, a preferred approach willinvolve the preparation of retroviral vectors which incorporate nucleicacid sequences encoding the desired construct, once introduced into thecells to be treated, preferably, these sequences are stably integratedinto the genome of the cell. One example of such of vector constructwould be a replication defective retrovirus, such as LNSX, LN or N2A,that is made infective by appropriate packaging, such as by GPtenvAM-12cells. Although the retrovirus would inhibit the growth of the tumor,the expression of the antisense construct in non-tumor cells would beessentially harmless where one prepares a retrovirus construct whichencode distinct antisense intron RNA in accordance with the presentinvention. In addition to retroviruses, it is contemplated that othervectors can be employed, including adenovirus, adeno-associated virus,or vaccinia viruses (Hermonat, et al., 1984; Karlsson, et al., 1985;Mason, et al., 1990).

[0038] The particular promoter that is employed to control theexpression of the antisense RNA in a vector construct is not believed tobe particularly crucial, so long as it is capable of expressing theantisense intron RNA in the targeted cell of a rate greater than 5 foldthat of the gene to be inhibited. Thus, where a human cell is targeted,it will be preferred to position the antisense RNA coding regionadjacent to and under the control of a promoter that is capable of beingexpressed in a human cell. Generally speaking, such a promoter mightinclude either a human cellular or viral promoter. While the β-actinpromoter is preferred the invention is by no means limited to thispromoter, and one may also mention by way of example promoters derivedfrom RSV, N2A, LN, LNSX, LNSN, SV40, LNCX or CMV (Miller, et al., 1989;Hamtzoponlos, et al., 1989).

[0039] The most preferred promoters will be those that are capable ofbeing expressed in a wide variety of histologic cell types, and which iscapable of continuously expressing the antisense RNA. A preferredexample is the β-actin promoter, because the promoter functionseffectively in human epithelial cells. Other examples of promotershaving a similar capability include RSV and SV40.

[0040] Where retroviral vectors are concerned, a more particular featureof the present invention is the general, overall design of preferredretroviral vector constructs. The most preferred vector design of thepresent invention takes into account the inventors' discovery that whena particular promoter, the β-actin promoter, is employed to driveexpression of a selected gene, and the expression construct ispositioned in an orientation that is opposite that of retroviraltranscription, there is a surprising increase in the relative expressionof the selected gene. Thus, generally speaking, retroviral constructs ofthe present invention can be said to include a gene expression unitwhich includes a selected gene under the control of a β-actin promoter,wherein the gene expression unit is positioned to effect transcriptionof the selected gene in an orientation opposite that of retroviraltranscription.

[0041] By “reverse orientation” or “opposite orientation” is meant thatthe orientation of transcription of the selected gene that is under thecontrol of the β-actin promoter is in the opposite direction from thedirection of transcription of the regular retroviral genes. Thus, forexample, where the vector includes a long terminal repeat (LTR), as domost retroviral vectors, the orientation of transcription of theselected gene will be opposite that of the LTR.

[0042] While the retroviral construct aspect of the present inventionconcerns the use of a β-actin promoter in reverse orientation, there isno limitation on the nature of the selected gene which one desires tohave expressed. Thus, the invention concerns the use ofantisense-encoding constructs as well as “sense” constructs that encodea desired protein.

[0043] Of particular importance is the inventors somewhat surprisingdiscovery that reversing the orientation of the genetic construct withrespect to the direction of transcription of the retroviral vectordramatically improves expression of the selected gene. This effect isdramatically illustrated in the context of K-ras antisense therapy (seeFIG. 9A and Example II below). In these studies, when the antisenseconstruct was expressed from a retroviral vector aligned in the samedirection of transcription as the retroviral LTR, the effect insuppressing target cells versus control cell growth was evident, buttarget cells growth was nonetheless observed by 7 days. In starkcontrast, no growth was observed after 7 days where the reverseorientation construct was employed.

[0044] The nature of the retroviral vector that is employed may dependupon the application that is envisioned. For clinical application, thereare several types of such vectors that have been found or proposed asapplicable, such as a Moloney murine leukemia virus vector, mousemammary tumor virus, or related retroviruses, or the like. The use ofthese vectors for clinical applicable rests upon the fact that they donot include active viral genes that could be considered harmful tohumans or animals and do not lead to the production of infective virusesupon infection. However, the invention is not limited in its scope toclinical applications, and for applications that do not contemplateclinical administration to humans or animals it is proposed thatvirtually any type of retrovirus can be employed.

[0045] Certain preferred vectors designed and employed by the presentinventors will include a second gene expression unit which includes asecond gene, such as a selectable marker gene, expressed from aretroviral long-term repeat. The presence of a selectable marker genesfacilitate the preparation of the vector by allowing the selection ofappropriate host cells from which the vector is prepared. The nature ofthe marker gene is not believed to be particularly crucial, so long asit does not produce a product that is harmful to the host cell, or tohumans or animals where clinical application is contemplated.

[0046] Where clinical application of retroviral vectors is contemplated,it will be necessary to prepare the vector and place it into apharmaceutical composition that is appropriate for the intendedapplication. This will entail generally preparing a pharmaceuticalcomposition that is essentially free of pyrogens, as well as any otherimpurities that could be harmful to humans or animals. One will alsogenerally desire to employ appropriate salts and buffers to render thevector stable and allow for vector uptake by target cells. Thepreparation of appropriate pharmaceutical retroviral compositions aregenerally well known, as are appropriate amounts, etc., of vectors to beemployed.

BRIEF DESCRIPTION OF THE DRAWINGS

[0047]FIG. 1A) The second exon of the K-ras gene was amplified fromgenomic DNA of H522, H322, Calu 1, H226, H460a and human placenta bypolymerase chain reaction (PCR), blotted onto a gene screen membrane andhybridized with ³²p end-labeled oligonucleotide probes. FIG. A1 showsthe presence of wild-type glutamine residue (CAA) at 61 codon in fivecell lines except H460a. The same blot was reprobed with ahistidine-specific mutated oligo probe (CAT) and only the H460a cellline PCR DNA hybridized (FIG. A2). The mutation was confirmed by directPCR DNA sequencing. Wild-type K-ras 61 codon sequence in human placenta(FIG. A3) was compared with the H460a cell line (FIG. A4).

[0048] B) A 2 kb genomic DNA segment from the K-ras oncogene wassubcloned into in Apr-1-neo vector in both a sense and antisenseorientation. A 2 kb Eco RI/Pst I fragment containing second and thirdexon sequences together with adjoining flanking intron sequences wasisolated from the SP6 vector (Oncogene Sciences) and Klenow enzyme wasused to make blunt ends. Apr-1-neo vector was digested with Bam HI andblunt end ligation was performed to obtain the Apr-1-neo AS or Apr-1-neoA constructs.

[0049] C) A southern blot analysis of the K-ras oncogene in H460a andH460a transfectants. Blots were probed with P32 nick translated 2 kb EcoR1/Pst1 insert DNA. 1) H460a, (2, 3) H460a transfected with Apr-1-neo SC₁#1 and C₂#1 (4, 5) H460a cells transfected with Apr-1-neo AS, C₃#32and C₂#32, respectively.

[0050] D) A northern blot analysis of sense and antisense K-ras RNA.1)H460a, (2, 3) Apr-1-neo S transfectants, (4, 5 ) Apr-1-neo AStransfected clones.

[0051] E, F) A Western blot analysis of K-ras specific p21-protein (1E)and total ras protein (1F) was performed using either pan ras or K-rasspecific monoclonal antibodies. 1) Calu-1 control cell line overexpressing K-ras specific protein. 2) H460a; 3) H460a Apr-1-neo S; 4, 5)H460a Apr-1-neo AS.

[0052] G) Map of plasmid pH β APr-1-neo

[0053]FIG. 2A) Schematic diagram of K-ras RNA synthesis. A segment ofras cDNA was amplified using oligonucleotide primers corresponding tothe 5′ region of first exon and 3′ of second exon (indicated by arrows)for RNA PCR analysis.

[0054] B) An RNA PCR analysis was done to compare the level of K-rasmessage in H460a and H460a transfectants. As a control, a portion of p53gene was co-amplified with p53 specific primer which served as aninternal control.

[0055] C, D) H-ras and N-ras specific amplimers were used to quantitateH-ras/N-ras RNA in the transfectants and parental cell lines. p53 geneamplification is shown as an internal control.

[0056]FIG. 3(A) In vitro growth curve. Cells were seeded at 10⁴cells/plate and grown for a seven day period. Cells were harvested andcounted in a hemocytometer at 24 h intervals. Growth curves for H460Aand H460A cells transfected with Apr-1-neo S vector do not show anysignificant difference, but H460A transfectants carrying Apr-1-neo-ASshowed growth inhibition (FIG. B). Female BALB/C nu/nu mice wereinjected with 10⁶ H460a cells subcutaneously in the left flank.Cross-sectional diameters of the external tumor were measured withoutknowledge of the cell group. Tumor volume was calculated by assuming aspherical shape with the average tumor diameter calculated as the squareroot of the product of cross-sectional diameters. Palpable tumors werefirst detected on day 15. Each point represents the mean ±SE. C3#32-AS(n=5), C3#1-S (n=5), H460a (n=3). C3#32-AS was compared to C3#1-S orH460a on days 20, 25, 30, 35 (p<0.05 by Wilcoxon's Test).

[0057]FIG. 4 Subcloning of β-actin K-ras antisense fragment in the LNSXretroviral vector. A 1.8-Kb genomic K-ras DNA segment with a 4-Kbβ-actin promoter in antisense orientation was subcloned into a 6-Kb LNSXretroviral vector using blunt (a) or Hind III linker (b) ligations intwo orientations.

[0058]FIG. 5 LNSX-antisense (a) retrovirus infection efficiency in H460acells. A. H460a cells 10⁵ in 6-well plates were infected once with 1 mlof each serial dilutions of retroviral stocks in the presence of 8 μg/mlpolybrene. Two days later, seeding equal numbers of H460a transducedcells into 300 μg/ml G418 selective medium or nonselective medium for10-14 d. Infection efficiency=(No. of colonies in G418 medium)/(No. ofcolonies in medium without G418). B. H460a cells 10⁴ in 12-well plateswere incubated with 0.5 ml LNSX-antisense (orientation a) retroviralstocks (Titer: 2×10⁶ CFU/ml). Polybrene 8 μg/ml was also added. Theinfections were done once each day for 1 to 7 d. Fresh medium andsupernatant were added at each time point. The infection efficiency wascalculated as for A.

[0059]FIG. 6 PCR analysis of transduced H460a cells. The genomic DNA ofH460a was extracted and amplified by PCR with neo 1 and neo 5oligonucleotide primers. The PCR products were electrophoresed on 2%ethidium bromide-stained agarose gel (A). The DNA was transferred ontonitrocellulose membranes and hybridized with ³²P-nick-translated neogene probe (B). Lane 1: molecular weight marker; Lane 2: H460a-antisenseLNSX (orientation a); Lane 3: H460a-antisense-LNSX (orientation b); Lane4: H460a-LNSX; Lane 5: parental H460a; Lane 6: LNSX vector plasmid DNA.

[0060]FIG. 7 Slot blot hybridization of poly(A+) RNA of H460a cells.Poly (A+) RNA was extracted, spotted onto nitrocellulose membranes (8μg, 4 μg, or 2 μg) and hybridized with ³²P-end-labeled 42 bp K-ras exon2 sense oligonucleotide probe (A). The filter was reprobed with a³²P-nick-translated β-actin probe to check for equal loading (B). Lane1: H460a-antisense-LNSX (orientation b); Lane 2: H460a-antisense-LNSX(orientation a); Lane 3: H460a-LNSX; Lane 4: H460a parental cells.

[0061]FIG. 8 Western blot analysis of ras p21 proteins in H460a cells.One hundred micrograms of protein was size fractionated by 12.5%SDS-polyacrylamide gel and electroblotted onto nitrocellulose membranes.K-ras-p21-specific (A) and pan-ras-specific monoclonal antibodies (B)were used, followed by HRP-labeled goat anti-mouse second antibody. Lane1: H460a parental cells; Lane 2: H460a-LNSX; Lane 3:H460a-antisense-LNSX (orientation b); Lane 4: H460a-antisense-LNSX(orientation a).

[0062]FIG. 9A. Growth curve of H460a cells in vitro. Cells 10³/well wereseeded in 12-well plates and grown for 7 days. Cells were harvested andcounted daily by trypan blue exclusion. B. Growth curve of MRC-9 cellsin vitro.

[0063]FIG. 10 Soft agarose colony formation of H460a cells. Cells 5×10⁴were mixed with 0.35% agarose in RPMI 1640 route medium and plated overa base layer of 0.7% agarose and culture medium hardened in 60-mmdishes. Colonies were counted 10-14 d later. A: Parental H460a; B:H460a-LNSX; C: H460a-antisense-LNSX (orientation a); D:H460a-antisense-LNSX (orientation b).

[0064]FIG. 11 Functional transduction efficiency of LNSX-AS-K-ras inH460a cells. Growth curves are shown for 10³ cells/well seeded in 12well plates. H460a cells were infected by incubation 0.5 m of viralsupernatant stock from either LNSX or LNSX-AS-K-ras (6×10⁶ CFU/ml) dailyfor 4 consecutive days in the presence of 8 μg/ml of polybrene. Theparental H460a cells served as a control. Cells were not selected withG418. Cells were counted daily. The mean ±SE is shown for 3 replicates.

[0065]FIG. 12 H460a cells were infected with LNSX-AS-K-ras by incubating10⁴ cells with 0.5 ml of viral stock (6×10⁶ CFU/ml) produced by thepackaging cell line GP+envAm12 in the presence of 8 μg/ml of polybrene.Infection was done daily for 1 to 7 days. Two days later cells wereplated in equal numbers into selective media containing 200μg/ml G418.Control H460a cells were plated at equal cell numbers to determinebaseline colony forming efficiency. The infection efficiency wasmeasured by determining the percent of the unselected colony numberformed by the G418 selected colonies.

[0066]FIG. 13 Growth curves are shown for 10⁴ cells/well seeded in 12well plates. H322a cells were infected by incubation 0.5 m of viralsupernatant stock from either LNSX, DC, LNSX-p53 or DC-p53 (10⁶ CFU/ml)on 2 consecutive days in the presence of 8 μg/ml of polybrene. Theparental H322a cells served as a control. Cells were not selected withG418. Cells were counted daily. The mean ±SE is shown for threereplicates.

[0067]FIG. 14 Growth curves are shown for 10⁴ cells/well seeded in 12well plates. H460a cells were infected by incubation 0.5 m of viralsupernatant stock from either LNSX, DC, LNSX-p53 or DC-p53 (10⁶ CFU/ml)in the presence of 8 μg/ml of polybrene. The parental H322a cells servedas a control. Cells were not selected with G418. Cells were counteddaily. The mean ±SE is shown for three replicates.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0068] Molecular Events in Lung Cancer Development

[0069] Lung cancer remains the leading cause of cancer deaths in theUnited States where it kills more than 140,000 people annually.Recently, age-adjusted mortality from lung cancer has surpassed thatfrom breast cancer in women. Although implementation ofsmoking-reduction programs has decreased the prevalence of smoking, lungcancer mortality rates will remain high well into the 21st century(Brown et al., 1988). Unfortunately, all current treatment modalities,including radiation therapy, surgery, and chemotherapy, have limitedeffectiveness. The rational development of new therapies for lung cancerwill depend on an understanding of the biology of lung cancer at themolecular level. Research carried out in the laboratories of the presentinventors has identified critical molecular events leading to NSCLCdevelopment and progression. The goal of this research is to directlymodify the cancer cell to eliminate the expression of gene productswhich are responsible for the maintenance or progression of themalignant phenotype or to restore in normal form deleted or mutated geneproducts that suppress the characteristics of the malignant phenotype.

[0070] The most common lung cancer histologies (80%) are grouped underthe term non-small-cell lung cancer (NSCLC) and include squamous,adenocarcinoma, and large-cell undifferentiated. Many of the currentdata on the molecular biology of lung cancer come from the study of themore uncommon small-cell lung cancer (SCLC). SCLC can be distinguishedfrom NSCLC by the neuroendocrine features of the cells; SCLC is veryresponsive to chemotherapy but recurs rapidly after treatment. NSCLCalso may serve as a model for other carcinogen-induced epithelialcancers. The approaches and observations developed in this study may beapplicable to other types of epithelial cancers.

[0071] Abundant evidence has accumulated that the process of malignanttransformation is mediated by a genetic paradigm (Bishop et al., 1991).The major lesions detected in cancer cells occur in dominant oncogenesand tumor suppressor genes. Dominant oncogenes have alterations in aclass of genes called proto-oncogenes, which participate in criticalnormal cell functions, including signal transduction and transcription.Primary modifications in the dominant oncogenes that confer the abilityto transform include point mutations, translocations, rearrangements,and amplification. Tumor suppressor genes appear to require homozygousloss of function, by mutation, deletion, or a combination of these fortransformation to occur. Some tumor suppressor genes appear to play arole in the governance of proliferation by regulation of transcription.It is possible that modification of the expression of dominant and tumorsuppressor oncogenes may influence certain characteristics of cells thatcontribute to the malignant phenotype.

[0072] Despite increasing knowledge of the mechanisms involved inoncogene-mediated transformation, little progress has occurred indeveloping therapeutic strategies that specifically target oncogenes andtheir products. Initially, research in this area was focused on dominantoncogenes, as these were the first to be characterized. DNA-mediatedgene transfer studies showed acquisition of the malignant phenotype bynormal cells following the transfer of DNA from malignant human tumors.Activated oncogenes of the ras family were identified by this techniquewith transfection of human DNA into mouse NIH 3T3 cells.

[0073] Oncogene Mutations in Lung Cancer

[0074] Activation of the K-ras oncogene occurs in human NSCLC (Santos etal., 1989, Shimizu et al., 1983). Recent studies using the polymerasechain reaction (PCR) and specific oligonucleotide hybridization showthat a third of NSCLC patients have ras family mutations (Rodenhuis etal., 1987; Rodenhuis et al., 1988). However, Reynolds and coworkers,using a sensitive NIH 3T3 cotransfection-nude mouse tumorigenicityassay, found that 12 of 14 (86%) lung tumor DNAs from smokers containedactivated proto-oncogenes related to the ras family (Reynolds et al.,1991). K-ras mutations occur primarily in adenocarcinomas, and the K-rasproto-oncogene has a point mutation in 30% to 40% of adenocarcinomas ofthe lung (Rodenhuis et al., 1987; Rodenhuis et al., 1988). Thus, aminimum of 32,000 patients per year are expected to developras-mutation-positive lung cancer. K-ras mutations are associated with ahistory of tobacco consumption and may contribute to tumor progression.

[0075] The p53 gene is the most frequently mutated gene yet identifiedin human cancers. It is mutated in over 50% of human NSCLC (Hollesteinet al., 1991). The p53 gene encodes a 375-amino-acid phosphoprotein thatcan form complexes with host proteins such as large-T antigen and E1B(Lane et al., 1990). Missense mutations are common for the p53 gene andare essential for the transforming ability of the oncogene. The wildtypep53 gene may directly suppress uncontrolled cell growth or indirectlyactivate genes that suppress this growth. Thus, absence or inactivationof wildtype p53 may contribute to transformation. However, some studiesindicate that the presence of mutant p53 may be necessary for fullexpression of the transforming potential of the gene. Mutations of p53are common in a wide spectrum of tumors (Bressac et al., 1990; Dolcettiet al., 1990; Rodrigues et al., 1990; Nigro et al., 1989); they occur inboth NSCLC and SCLC cell lines and fresh tumors (Nigro et al., 1989;Takahashi et al., 1989).

[0076] Options for specific targeting of oncogenes include inhibition ofexpression of a dominant gene or replacement of a deleted or mutatedtumor suppressor gene. Progress in the understanding of the criticalgenes involved in tumor development and in technology for altering geneexpression logically led to our studies of techniques for achievingthese options. Initially, a model for specific inhibition of K-ras wasdeveloped. We chose to work with K-ras because of the applicability ofthe findings to a large number of tumors, because of our previous workwith K-ras, and because information on the genetic organization andsequence of the ras gene family was readily available. Advances inantisense and retroviral gene transfer technology suggested thatapplication of these techniques may mediate specific inhibition ofoncogene expression.

[0077] Antisense mRNA, which is precisely complementary to thecorresponding sense mRNA, inhibits translation. The mechanisms for thisinhibition have not been completely defined but include inhibition oftranslation by ribosomes, degradation of sense-antisense duplexes byenzymes, and failure of export from the nucleus. Thus, specifictargeting of a gene in a multigene family could occur if it possessedunique sequences in a region amenable to antisense inhibition, such asan initiation codon or splice site.

[0078] The working hypothesis that was developed by the inventors isthat reversal of a single altered genetic event in the cancer cell canpotentially reverse critical features of the malignant phenotype of thatcell. This finding has important therapeutic implications. Cancer cellshave multiple genetic alterations. Therapy directed toward oncogeneswill be practical only if therapeutic effects occur with targeting ofone or two genes. It is unlikely that any therapy targeting oncogenes ortheir products will be absolutely specific for cancer cells. If othergenes can compensate for loss of normal function by a specific oncogenemediated by an antisense construct, the harmful effects of the therapywill be reduced.

[0079] Studies from the inventors' laboratory indicate that reversal ofa single genetic alteration has profound effects on the growth andtumorigenicity of lung cancer cells (Mukopadhyay et al., 1990;Mukopadhyay et al., 1991). Additional support for this concept comesfrom a recent study by Soriano and coworkers (Soriano et al., 1991) inwhich transgenic mice were created that lacked a functional c-srcproto-oncogene. The resulting developmental defect in the mice wasosteopetrosis. The ubiquity of c-src, its high degree of conservationamong species, and its role in mitosis suggest that inactivation wouldbe lethal, but this was not the case; viable mice were recovered. Apossible explanation is that other closely related nonreceptor tyrosinekinases such as yes and fyn can compensate for loss of c-src.Introduction of a single copy of a wildtype tumor suppressor gene intonormal cells would be unlikely to have adverse effects if it occurredduring therapy directed at replacing inactivated tumor suppressor genesin cancer cells.

[0080] Preliminary data on transfection of an antisense K-ras expressionvector indicated that inhibition of expression of a single oncogenereduced the growth rate of cancer cells and tumorigenicity in nu/numice. However, transfected cells retained viability, as did cells withno endogenous K-ras mutation that were also transfected with theconstruct. The wtp53 appears dominant over the mutant gene and willselect against proliferation when transfected into cells with the mutantgene (Mukhopadhyay et al., 1991; Chen et al., 1990). Normal expressionof the transfected wtp53 does not affect the growth of cells withendogenous wtp53. Thus, such constructs might be taken up by normalcells without adverse effects.

[0081] Treatment Protocol Development

[0082] The inventors have developed a protocol for the treatment oftumors susceptible to either wtp53 or antisense K-ras gene therapy. Thisprotocol focuses regional delivery of the two gene constructs, antisenseK-ras and wtp53, to lung cancer cells in patients with unresectableobstructing endobronchial cancers. The efficiency of delivery and geneexpression will be evaluated both in lung cancer cells and in normalcells in vivo. This is of importance for the design of constructs thatmay be useful therapeutically. The effects of these constructs onclinical progression of the cancer will be studied.

[0083] It is proposed that these approaches will lead to cancer therapybased on direct alteration of gene expression in cancer cells. Currenttherapy relies on attempts to kill or remove the last cancer cell.However, tumor cell dormancy is an established phenomenon makingeffective killing highly unlikely. Although inhibition of expression ofsome oncogenes may be lethal to the cancer cell, in some cases cellreplication will slow or cease, thus rendering these cancers clinicallydormant. Even if absolute specificity is not achieved, single oncogenesmay still be important targets, because it is likely that adverseeffects to normal cells will be minimal.

[0084] Natural History of Locally Unresectable NSCLC

[0085] Patients with NSCLC will die of their cancer in 86% of cases.Regional delivery of gene constructs to areas at risk for development ofcancer has important implications for both prevention and therapy.Failure of therapy at the primary tumor site is a significant problem(Humphrey et al., 1990; Perez et al., 1990). Of the 161,000 patientsnewly diagnosed with lung cancer in 1991, 45,080 will undergo surgicalresection. Local recurrence as the first site of failure will occur in9,000 of those patients. Of the remaining patients, 52% will havelocalized tumors. However, 38% of these patients will have localfailures following radiation therapy (22,900). Thus, 31,900 patients peryear could benefit from improved local-regional therapy. Patients withunresectable obstructing NSCLC that is resistant to radiation therapy orwho have coexisting metastases have a median survival of 6 months orless (Komaki et al., 1992).

[0086] Measure of Disease Activity

[0087] The ultimate goal of this therapy is to halt or reverse themanifestations of the disease. The efficacy of therapy in this group ofpatients will be measured by determining length of patient survival,length of time the affected lobe of the lung remains aerated, andreduction in measurable endobronchial tumor. There is no curativetherapy for this stage of disease and thus the outcome is predictableenough to allow for an assessment of the results of gene therapy.

[0088] Anticipated Effect of Protocol Treatment

[0089] It is anticipated that the uptake of the retroviral constructs byproliferating NSCLC cells will decrease the rate of proliferation ofthese cells. This would increase the length of time the affected lungwould remain expanded, prevent regrowth of the endobronchial tumor, andprolong the patient's survival.

[0090] Alternative Therapies

[0091] Patients with unresectable endobronchial tumor recurrence that ispartially or completely obstructing the airway and that have failed orare unable to receive external beam radiotherapy will be considered forthis protocol. Existing therapies for this condition offer onlyshort-term palliation. Most patients have recurred despite external beamradiotherapy. It may be possible to insert a brachytherapy catheter andadminister additional radiotherapy. Patients receiving this treatmenthave a median survival of 6 months (Komaki et al., 1992). Patientsfailing brachytherapy would also be eligible to receive gene therapy.Tumor can be removed from the airway with the laser or biopsy forceps.This can be done in conjunction with injection of the retroviralconstruct thus decreasing the volume that must be injected. Theadministration of the retroviral constructs would not preclude thepatient from receiving other palliative therapy if the-tumor progresses.

[0092] Antisense Embodiments

[0093] As noted above, where one contemplates employing an antisenseapproach to selectively inhibit one of a family of genes, it will beparticularly advantageous to include within the construct regionsencoding an antisense intron region complementary to an intron unique tothe target transcript. In such circumstances, the present invention willbe generally applicable to the down-regulation of any gene whichcomprises a distinct intron region, particularly those oncogenes whichare members of family wherein one desires to leave unaltered theexpression of other family members.

[0094] The present invention will have particular application to theselective inhibition of ras gene expression. For example, in the case ofras gene tumorigenesis, only one of the various ras gene family membersundergoes mutation-based protooncogene activation. The remaining,non-activated ras gene family member(s) serve useful cellular biologicalfunctions and are apparently required for normal cellular function.Thus, it is desirable to specifically down-regulate the activated rasgene product, while leaving essentially unaffected, the non-activatedras gene counterparts. Thus, the present invention will have aparticular application in the context of preferentially controlling rasgene expressing.

[0095] While this aspect of the invention is exemplified in terms of thecontrol of ras gene expression, there is, of course, no reason why thepresent invention will not be similarly applicable to other genes andgene families, in light of the disclosure herein and the generalknowledge and skill in the art.

[0096] Generally speaking, to practice the antisense/intron aspects ofthe present invention in the context of the ras gene system it will befirst important to determine which of the various ras genes is involvedin the oncogenic process to be retarded. This is a fairlystraightforward undertaking, and involves generally that one firstobtain cells which are expressing the activated ras gene product. Todetermine the nature of the activation, one then simply extracts DNA,amplifies the specific sequences of interest (see Table 1 below), andshows the presence or absence of the mutation by either direct sequenceanalysis or specific hybridization with a known oligonucleotidesequence.

[0097] After the particular activated ras gene has been identified, anappropriate intron region is then selected for constructing theantisense construct. The most appropriate introns are those which havelittle or no homology to other known genes. In general, it will bepreferable to identify an appropriate intron structure for use inconnection with the present invention an analysis of the nucleicsequence of the intron, and comparison with selected that of introns ofother family members or related genes. The best choice of introns willbe those having 1) a different length from corresponding introns andsimilar location in other members of the gene family, and 2) little orno sequence homology with the introns of the other members.

[0098] An alternative, and sometimes simpler method to identify distinctintrons involves a comparison of sequence homologies can be asertainedby cross-hybridization of introns from one family member with those ofother genes.

[0099] In any event, representative methods for cloning ras genescorresponding to the N-ras, K-ras and H-ras genes, have been describedin the literature (McGrath, et al., 1983; Shimizu, et al., 1983;Yamamoto, et al., 1985; Kraus, et al., 1984). These teachings shouldprovide those of skill in the art with adequate direction where oneseeks to obtain sequences corresponding to the various ras gene intron.

[0100] A preferred method for cloning intron sequences is through theapplication of PCR-amplified cloning. In this relatively well knowntechnique, one employs oligonucleotide primers which allow the specificamplification of the desired intron region. The primer itselfcorresponds to exon sequences, in that these sequences will most likelybe generally available in the scientific literature for the particularapplication envisioned. Of course, where the intron sequences are known,computer assisted comparisons may be carried out to identify distinctregions, and appropriate PCR primers designed accordingly.

[0101] Recombinant clones which incorporate intron DNA are readilyachieved through the PCR amplification of the distinct desires regionusing primers, e.g., that border the region, incorporating the amplifiedDNA into a recombinant clone, and selecting recombinant clones whichhave received the intron DNA-bearing clones. The intron DNA containingclones are then purified, and, preferably, the cloned DNA sequencedsufficiently to ensure that it contains the desired sequences.

[0102] Intron DNA is then removed from the vector employed for intronDNA cloning, and employed in the construction of appropriate antisensevectors. This will entail, of course, placing the intron DNA in anantisense direction behind an appropriate promoter and positioned so asto bring the expression of the antisense intron under control of thepromoter.

[0103] When selecting primers for intron sequence amplification, onewill typically desire to employ primers such that at least 50 andpreferably 100-200, nucleotides of the intron are amplified and therebycloned. In general, it is believed that the larger the distinctantisense intron region is, the better able it will be to selectivelydown-regulate the targeted gene. Furthermore, it is believed thatparticular advantages will be realized through the selection of intronregions which include intron/exon boundaries, or simply just the intronside of the intron/exon boundaries. The reason for this is that RNAprocessing takes place at the intron/exon boundary of the RNA and it isbelieved that the antisense intron DNA will have its greatest effectwhen targeted to this junction.

[0104] The particular vector which one employs for introduction ofantisense intron coding sequences is not believed to be particularlycrucial to the practice of the present invention, so long as the vectoris capable of introducing the nucleic acid coding sequences into thegenome of the targeted cell in a relatively stable fashion. By way ofillustration, but not limitation, one can mention the following vectors,including N2A, LN, LNSX, Adenovirus and Adeno-associated virus.

[0105] The most preferred vector construct for targeting cells is theLNSX retroviral vector. This vector is based on the N2 vector, whichcontains the extended packaging signal that allows for the production ofthe vector at a high titer. This vector was modified by inserting a stopcodon in place of the Pr65 gag start codon to prevent synthesis of Pr 65gag, and by replacing the upstream region of the vector with thehomologous region from Moloney murine sarcoma virus. These alterationsprevent synthesis of viral proteins from the vector. Splicing is notrequired for efficient neo-protein expression. The neo gene is expressedfrom the upstream LTR promoter.

[0106] The following examples are included to provide actual workingprotocols which the inventors have developed or adopted for carrying outpreferred embodiments of the invention. Those of skill in the art willreadily appreciate that many of the techniques employed in the followingexamples are illustrative of standard laboratory practices, which havebeen found by the inventors to work well in the practice of theinvention. It will, however, be apparent to those of skill in the art,in light of the following examples, that numerous materials and/ormodifications and procedures and nevertheless achieve a useful result.

EXAMPLE I Specific Inhibition of K-RAS Expression and Tumorigenicity ofLung Cancer Cells by Antisense RNA

[0107] A. Introduction

[0108] A wide spectrum of human cancers harbor ras genes activated by asingle point mutation (Barbacid, 1987; Rodenhuis, et al., 1987; Bos,1989; Rodenhuis, et al., 1990; Mabry, et al., 1988; Santos, et al.,1984; Taya, et al., 1984; Cline, et al., 1987; Feig, et al., 1984;Vogelstein, et al., 1988; Kumar, et al., 1990). Despite considerableknowledge of the structural aspects of the ras gene product, thefunctional role in physiological and pathological processes remainselusive (Barbacid, 1987). Cellular location and structural andbiochemical similarities to G proteins suggest that ras gene productsare involved in signal transduction (Bos, et al., 1987; Hurley, et al.,1984). The present example describes the preparation and use of anantisense RNA construct to block selectively the production of themutated protein in the human non-small cell lung cancer (NSCLC) cellline NCI-H460A. The direct contribution of the mutated p21 protein tothe malignant phenotype was also examined.

[0109] B. Materials and Methods

[0110] H460, H322, H226, H522 non-small cell lung cancer (NSCLC) celllines were generously provided by Drs. J. D. Minna, A. F. Gazdar, NCINaval Medical oncology Branch, Bethesda, Md. All cell lines were grownin regular RPMI medium, 5% FCS, in routine culture.

[0111] 1. Plasmid Construction

[0112] A 2-kb genomic DNA fragment from the K-ras proto-oncogene wassubcloned into an Apr-1-neo vector in both sense and antisenseorientation. A 2-kb Eco RI/Pst I fragment containing second and thirdexon sequences together with adjoining flanking intron sequences wasisolated from the SP6 vector (Oncogene Sciences) and Klenow enzyme wasused to make blunt ends. Apr-1-neo vector was digested with Bam HI andblunt end ligation was performed to obtain the Apr-1-neo AS or Apr-1-neoA constructs.

[0113] 2. DNA Transfections

[0114] H460a or H322a cells were electroporated with 10 μg of Apr-1-neoAS or Apr-1-neo S plasmid DNA. Forty-eight hours after transfection G418was added into the medium at a concentration of 300 μg/ml for H460a and200 μg/ml for H322a. Individual colonies were picked up and grown inculture for further analysis.

[0115] 3. Southern blot analysis

[0116] High molecular weight DNA was isolated and digested with Eco R1(Boehringer-Mannheim) (20 μg), and electrophoresed in 0.8% agarose gel,transferred onto a Gene Screen membrane (NEN) and hybridized with a P³²nick translated 2kb genomic K-ras DNA probe.

[0117] 4. Measurement of RNA Expression

[0118] Total cellular RNA was isolated from the cell lines (Chomczymsky,et al., 1987). Twenty microgram of total RNA was size fractionated inMOPS/formaldehyde gel, transferred onto a Gene Screen membrane andprocessed for hybridization with riboprobes. A 302 bp genomic DNA of theK-ras gene was amplified by PCR spanning the third exon and intronsequences and was subcloned into a bluescript vector. In vitro S and ASRNA probes were synthesized using either a T7 or T3 promotor.

[0119] 5. Polymerase Chain Reaction

[0120] Polymerase chain reactions were performed as previously describedusing Taq 1 DNA polymerase (Saiki, et al., 1985). Oligonucleotideprimers corresponding to region the 5′ and 3′ regions of codons 12 and61 of human K-ras, H-ras, and N-ras genes were synthesized. Twomicrograms of genomic DNA was subjected to 35 cycles of amplification.DNA sequences of oligonucleotide primers used for PCR amplification arelisted below in Table 1. TABLE 1 Primers Sequence Target KA61 5′ TTC CTACAG GAA GCA AGT AGT A 3′ K-ras 2nd exon KB61 5′     ACA CAA AGA AAG CCCDCC CCA 3′ KA12 5′ GAC TGA ATA TAA SCT TGT GG 3′ K-ras 1st & 2nd exonKB61 5′ ACA CAA AGA AAG CCC DCC CCA 3′ HA12 5′ GAC GGA ATA TAA GCT GGTGG 3′ H-ras 1st & 2nd exon HB61 5′ CGC ATG TAC TGG TCC CGC AT 3′ NA125′ GSC TGA GTA CAA ACT GGT GG 3′ N-ras 1st & 2nd exon NB61 5′ ATA CACAGA GGA AGC CTT CG 3′

[0121] 6. Slot Blot Oligonucleotide Hybridization p PCR amplified DNAsamples (12.5, 25, 50 ng) were blotted onto a Gene Screen membrane usinga slot blot apparatus (Schleicher & Schuell). The filters wereprehybridized and hybridized at 55° C. in 6×SSC, 5×Denhardts and 100μg/ml of salmon sperm DNA for 2 h. Filters were washed twice in 6×SSPEat room temperature and once for 30 mins at 58° C. Finally, blots werewashed for 5 mins at 64° C. The filters were exposed to x-ray film for12-24 h at -80° C.

[0122] 7. Direct Sequencing of PCR Amplified DNAs

[0123] PCR DNA corresponding to the second exon was purified in 8%polyacrylamide gel. A single DNA band was excised and purified DNA wasused for asymmetric amplification in 100 μl of PCR reaction mixture. One(KA 61) amplimer was added to this mixture. After 20 cycles,single-stranded DNA was purified through gene clean (Bio 101) and DNAwas eluted in 15 μl of water. Four microliters of DNA were mixed with 4μl of 10×Taq 1 buffer and 1 μl (10 pmol) of a second amplimer (KB 61)was used as a sequencing primer and DNA was sequenced using a Sequenasekit.

[0124] 8. RNA PCR Analysis

[0125] cDNA synthesis was carried out in a total volume of 20 μlcontaining 5 μg of total RNA and oligo (dT) as a primer (Becker-Andre,et al., 1989). A portion of the cDNA corresponding to the first andsecond exons was amplified to monitor the level of endogenous K-ras mRNA(FIG. 2A) using KA12 and KB61 amplimiers. Denaturation, annealing, andextension were done at 92° C. for 1 min, 51° C. for 1 min and 74° C. for1 min, respectively. However, annealing temperatures for N-ras and H-raswere 44° C. and 42° C., respectively. In addition, two amplimers werealso used in the same reaction mixture to amplify a 118-bp fragment ofthe p53 gene as an internal control. PCR products were eithertransferred onto a membrane and hybridized with ³²p labelled cDNA probeor alternatively, there were directly labelled during the last cycle ofamplification by adding 1 uCi of ³²p dCTP. The labelled PCR productswere loaded on an 8% nondenaturing polyacrylamide gel. The gel wasphotographed after ethidium bromide staining, dried, and exposed tox-ray film overnight at−80° C.

[0126] 9. Western blot analysis of RAS protein

[0127] Protein extracts were prepared by lysing cell in TBS (10 mM TRISph 7.5, 100 mM Nacl, 1 mM PMSF 1% NP40, 1% deoxycholate. The extractswere cleaned by centrifugation at 10,000×g for 1 h. The proteinconcentration of the supernatant was calculated spectrophotometrically.Five hundred micrograms of protein were size fractionated in 12.55% SDSpolyacrylamide gel and electroblotted onto nitrocellulose membranes. Rasspecific p21 protein was detected using either K-ras or pan ras specificmonoclonal antibody (Oncogene Sciences) followed by ¹²⁵I-labelled goatanti-mouse second antibody.

[0128] 10. Tumorgenicity in Nude Mice

[0129] The tumorigenicity of these cell lines was examined bysubcutaneous inoculation of 10⁵ (FIG. 3B) and 10⁶ cells in nu/nu mice.Each cell line was injected into 5 animals. Tumors were measured withlinear calipers in 2 orthogonal directions by the same observer.

[0130] C. Results and Discussion

[0131] Segments of the K-ras gene containing first and second exons wereamplified from a number of NSCLC cell line DNAs by polymerase chainreaction (Saiki, et al, 1985) and subsequently hybridized with a set of³²p-labelled oligonucleotide probes (FIG. 1A-1 & 2). Mutations wereconfirmed by a direct PCR DNA sequencing method. A homozygous mutationat codon 61 was detected in the NCI-H460A large cell undifferentiatedNSCLC cell line with a normal glutamine residue (CAA) substituted byhistidine (CAT). This cell line is highly tumorigenic in nude mice.

[0132] A recombinant plasmid clone was constructed using a wild-type 2kb K-ras genomic DNA segment carrying second and third exons togetherwith flanking intron sequences subcloned into an Apr-1-neo expressionvector (Gunning, et al., 1984) in the antisense orientation (AS; FIG.1G). Sense orientation (S) plasmid constructs were used as a control(FIG. 1B). AS or S K-ras RNA synthesis was accomplished by transfectingH460a cells, a cloned derivative of the NCI-H460A cell line, withApr-1-neo AS or Apr-1-neo S constructs by electroporation. The β-actinpromoter of the vector was constitutively capable of directing thesynthesis of RNA from the inserted DNA. The Apr-1-neo vector offeredsuitable G418 marker gene expression for selection of the transfectants.

[0133] Individual G418 resistant colonies were selected and grown inculture for further analysis. Stable integration of the plasmid DNA inthe transfectants was examined by Southern hybridization with a 2 kb DNAinsert from the original plasmid clone as a probe (FIG. 1C). Thesouthern blot analysis showed a single 3 kb Eco RI band corresponding tothe endogenous K-ras gene in the parental H460a cell line, butadditional bands were observed in the individual clones indicatingsingle or multiple copy inserts.

[0134] The extent of stable AS RNA expression and its effect on theendogenous K-ras mRNA level was investigated. Total RNA was extractedfrom subconfluent, growing cultures (Gunning, et al., 1987). Thepresence of AS and S RNA was detected by northern blot hybridizationusing either an S or AS RNA probe synthesized in vitro from a Bluescriptvector carrying a 302 bp K-ras DNA insert corresponding to the thirdexon and part of the intron sequences (FIG. 1D). Interestingly, theclones carrying the Apr-1-neo AS vector show one RNA band at about 1.5kb, but the cells carrying the S construct show two RNA species. Thereason for this is unknown, but the possibility exists that the RNAsynthesized from the genomic DNA under control of the β-actin promotercould be processed in vivo. However, no corresponding hybridization bandwas detected in H460a cells, which indicated a significantly higherlevel of K-ras RNA was synthesized under the β-actin promoter.

[0135] Next, the p21 protein level in these transfectants was analyzedby western blot analysis (FIG. 1E, F). A K-ras-specific p21 monoclonalantibody (Oncogene Science) was used to determine the level of K-rasprotein in transfectants, parental H460a cells, and Calu-1 cells, whichhave a high level of K-ras gene expression (FIG. 1E). Western blotanalysis showed a 95% reduction in K-ras p21 protein synthesis in theclones expressing the AS RNA, while parental cells, S K-ras clones, andCalu-1 cells showed a significant level of K-ras p21 protein. Theseresults indicate that AS RNA can effectively block the synthesis ofK-ras specific protein. Since members of the ras gene family share agreat deal of sequence homology and code for a similar p21 ras protein,we examined the total ras protein product in these clones was examinedusing a PAN ras monoclonal antibody (New England Nuclear) to determinewhether a reduced level of K-ras protein reflects any change in H-rasand N-ras p21 protein synthesis (FIG. 1F). Western blot analysisrevealed only a slight decrease in overall ras protein level in allclones containing Apr-1-neo-AS, as compared to 460a parental cells.

[0136] The effect of AS RNA on the specific production of matureendogenous K-ras mRNA was analyzed by cDNA PCR (FIG. 2). cDNAsynthesized from the total RNA (Chomczymsky, et al., 1987) was subjectedto PCR amplification using amplimers corresponding to the 5′-end of thefirst exon and the 3′-end of the second exon (FIG. 2A). Because the ASRNA was generated only from a second and third exon of the K-ras gene,PCR amplified cDNA represented the level of endogenous K-ras mRNA. A246-bp amplified DNA fragment was labelled by ³²P dCTP and subsequentlyanalyzed by polyacrylamide gel electrophoresis. In addition, a 118-bpsegment of endogenous p53 cDNA was co-amplified in the same reactionmixture using p53 specific amplimers to serve as an internal control forthe PCR.

[0137] Results showed that H460a cells, clones expressing S RNA, and theCalu-1 cell line expressed K-ras mRNA, as evidenced by the presence of ahigh level of amplification of the 246-bp cDNA product (FIG. 2B). H460aclones expressing AS RNA showed very little amplification, and cellularK-ras mRNA synthesis appeared to be completely inhibited (FIG. 2B, lanes5 and 6). In contrast, the endogenous p53 expression remainedunaffected. This prompted us to investigate the level of expression forother ras genes in these clones. We employed the same cDNA PCRmethodology to analyze the N-ras and H-ras mRNA level using N-ras andH-ras-specific oligonucleotides as amplimers. A steady state level ofH-ras and N-ras gene expression was observed, but no obvious changeeither in Apr-1-neo AS or Apr-1-neo S transfectants was noticed (FIG.2C, D). The p53 gene expression serving as a control in theseexperiments remained unaffected. Thus, inhibition of K-ras expression byour AS RNA construct is specific.

[0138] H460a clones expressing AS K-ras RNA continued to grow inculture. However, H460a Apr-1-neo AS transfectants showed a three-foldreduction in growth, compared to the H460a Apr-1-neo-S transfectants andthe parental H460a cells (FIG. 3A). The H322 NSCLC cell lung cancer cellline has wild-type ras family genes. H322 Apr-1-neo AS and Apr-1-neo Stransfectants had identical growth characteristics, indicating thatinhibition of wild-type K-ras is not sufficient to alter tumor cellgrowth rate. These results together indicate that the presence of senseK-ras RNA did not alter the growth kinetics of H460a cells. However, themarked growth retardation of the K-ras Apr-1-neo-AS transfectantssuggests that the mutated p21 protein contributes to the faster growthrate of these cells.

[0139] The tumorigenicity of cell lines expressing AS RNA was assessedby subcutaneous injection of 10⁵ and 10⁶ cells in nu/nu mice.Subcutaneous inoculation of H460a cells at both doses led to theformation of tumors in 15 days in all mice (3 to 5 mice per group in 3separate experiments). No tumor developed in mice injected with 10⁵cells for both clones of H460a AS cells during 120 days of observationin a total of ten mice, whereas all mice receiving H460a cells developedtumors. When the inoculum was increased to 10⁶ cells, tumors grew in allmice, but the tumors in mice receiving AS clones grew at a slower ratethan H460a cells or the S control (FIG. 3B). Tumors were excised andanalyzed for K-ras expression by cDNA-PCR. K-ras expression was notdetected in tumors arising from injection of AS clones but was presentin S clones and H460a tumors.

[0140] The above experiments indicate that in H460a cells engineered tosynthesize AS K-ras RNA, the level of K-ras mRNA and K-ras p21 proteinare effectively down regulated. Reduction in the expression of K-rasmutated gene reproducibly reduced the rate of tumor growth in nu/numice. Our studies show that a construct can be made that distinguishesamong members of the ras family. Previous studies with ASoligonucleotides showed inhibition of p21 expression which led to celldeath (Brown, et al., 1989; Debus, et al., 1990). Our data indicate thatAS RNA generated from the genomic DNA of the K-ras gene can specificallyinhibit K-ras expression. In our model inhibition of activated K-rasreduced the growth rate of the H460a cells. However, there was no effecton cell viability or continued growth in culture. This suggests thatredundancy in p21 expression may compensate for absence of expression byone member of this family so that functions essential for maintenance ofcell viability are preserved. However, tumorigenicity was maintained inthe absence of activated K-ras expression although the rate of tumorgrowth was diminished. We hypothesize that in human NSCLC, ras mutationsconfer a growth advantage to the malignant cell.

EXAMPLE II Retroviral Vector-mediated Transduction of K-ras AntisenseRNA Into Human Lung Cancer Cells Inhibits Expression of the MalignantPhenotype

[0141] In overview, the present example illustrates a retroviral vectorsystem that was developed by the inventors to efficiently transduceK-ras antisense constructs into human cancer cells. The 1.8-Kb K-rasgene fragment DNA in antisense (AS) orientation to a β-actin promoterwas inserted into retroviral vector LNSX in two different orientations.The constructs were transfected into amphotropic packaging cell lineGP+envAm12 followed by alternating infection between the ecotropicpackaging cell line Ψ2 and GP+envAm12. Titers up to 9×10⁶ CFU/ml wereachieved without detectable replication-competent virus being produced.The human large cell lung carcinoma cell line H460a, which has ahomozygous codon 61 K-ras mutation, was transduced, and a transductionefficiency of 95% was obtained after 5 to 7 repeated infections.

[0142] DNA polymerase chain reaction analysis showed that the retroviralconstruct was integrated into the genome of H460a cells. K-ras antisenseRNA expression was detected in the cells by slot blot hybridization witha specific oligonucleotide probe. Translation of the mutated K-ras p21protein RNA was specifically inhibited, whereas expression of other p21species was unchanged. Proliferation of H460a cells was suppressedtenfold following transduction by LNSX-AS-K-ras. Colony formation insoft agarose and tumorigenicity in an orthotopic nu/nu mouse model weredramatically decreased in H460a cells expressing antisense K-ras.

[0143] A. Materials and Methods

[0144] 1. Cells and Culture Conditions

[0145] NIH-3T3 cells, the human fibroblast cell line MRC-9, andecotropic retrovirus packaging cell line Ψ2 (Mann et al., 1983) weregrown in Dulbecco-modified Eagle's Medium (DMEM; GIBCO) with a highglucose content (4.5 g/l) supplemented with 10% fetal bovine serum(Sigma Chemical Co.). The amphotropic retrovirus packaging cell lineGP+envAm12 [(Markowitz et al., 1988); a gift from Dr. Arthur Bank] wasgrown in DMEM with high glucose; 10% newborn calf serum; 15 μg/mlhypoxanthine, 250 μg/ml xanthine, and 25 μg/ml mycophenolic acid (HXMmedium); and 200 μg/ml hygromycin B (Sigma Chemical Co.). Non-small celllung cancer cell (NSCLC) line H460a was maintained in RPMI 1640 mediumwith 5% fetal bovine serum (Mukhopadhyay et al., 1991). All cells werealso supplemented with 2 Mm L-glutamine and antibiotics. The H460a wasestablished in culture from a human large cell undifferentiatednon-small cell lung cancer. This cell line has a homozygous codon 61K-ras mutation (Mukhopadhyay et al., 1991). The MRC-9 cell line has noevidence of mutations at codon 12 or 61 of the K-ras gene by singlestrand conformation polymorphism (SSCP) analysis and chain terminationsequencing.

[0146] 2. Retroviral Vector Construction

[0147] Retroviral vector LNSX contains the selectable neo gene and aunique cloning site for cDNA insertion. The neo gene is expressed fromthe retroviral long-term repeat (LTR), and the inserted gene has thesimian virus 40 (SV4 early promoter (Miller et al., 1989). A recombinantplasmid clone was constructed using a wild-type 2-Kb genomic DNA segmentcarrying second and third exons together with flanking intron sequencessubcloned into an Apr-1-neo expression vector in the antisenseorientation with a β-actin promoter (Mukhopadhyay et al., 1991). The5.8-Kb EcoR I/Nde I fragment of β-actin K-ras antisense was isolatedfrom this plasmid, and Klenow enzyme was used to blunt the ends. Toobtain the recombinant constructs in two different orientations (a andb) relative to the SV40 promoter (FIG. 4A), the LNSX retroviral vectorwas digested with Stu I (orientation a) or Hind III (orientation b) andblunt end ligation or Hind III linker ligation was performed. E. colibacteria were transformed by this recombinant plasmid DNA, and cloneswere screened by enzyme analysis. Southern hybridization with the 1.8-Kb³²P-nick-translated genomic K-ras DNA fragment probe was used to confirmthe construction of the positive clones using the followinghybridization condition: 6×SSC, 10×Denhart's solution, 0.1% sodiumdodecyl sulfate (SDS), 100 μg/ml salmon sperm DNA, and 25 Mm NaH₂PO₄ for2.5 h at 65° C.

[0148] 3. Virus Production and Infection Efficiency

[0149] Amphotropic packaging cell line GP+envAm12 was transfected withrecombinant β-actin K-ras antisense LNSX plasmid DNA by the calciumphosphate co-precipitation method (Graham et al., 1973). Forty-eighthours later, the transfected cells were placed in medium containing G418(400 μg/ml). Colonies of “producer cells” were selected 10-14 d laterand expanded into large cultures.

[0150] The viral titer was tested by infecting NIH-3T3 cells. Plates (60mm) were each seeded with 5×10⁵ NIH-3T3 cells. After 24 h, the medium onthese plates was replaced with 1 ml of serial dilutions of mediumconditioned for 24 h by confluent cultures of producer cells. Polybrenewas added to a final concentration of 8 μg/ml. The cells were incubated2-4 h and then 4 ml of fresh medium was added. Forty-eight h after theinfection, the infected cells were trypsinized and replated onto 100-mmtissue-culture dishes in medium containing 400 82 g/ml G418. Coloniescould be counted 10-14 d later.

[0151] The high-titer GP+envAm12 cells transfected by β-actin K-rasantisense LNSX (orientation a) were mixed with ecotropic packaging cellline Ψ2 at a ratio of 1:1. A total of 5×10⁵ cells from this mixture wasseeded onto 100-mm plates and passaged continuously for 1 month. Thesecells were then selected by HXM medium (containing 200 μg/ml hygromycinB and 400 μg/ml G418) for 10-14 d. The amplification of retrovirusproduction was tested by infecting NIH-3T3 cells. Supernatants fromNIH-3T3 cells infected by GP+envAm12-producing cells and selected with400 μg/ml G418 for 10-14 d (short-term assay) or passaged continuouslyfor 1 month without G418 selection (long-term assay) were used to infectfresh NIH-3T3 cells to detect the existence of replication-competentretrovirus.

[0152] NSCLC cell line H460a was infected once by incubating 10⁵ cellsin 6-well plates with 1 ml of each serial dilution (1:1, 1:10, 1:100,1:1000) of recombinant LNSX-antisense (orientation a) retroviral stockin the presence of 8 μg/ml polybrene. In another assay, 10⁴ H460a cellswere incubated with 0.5 ml LNSX-antisense (orientation a) retroviralstock (virus titer: 2×10⁶ CFU/ml) in 12-well plates, and 8 μg/mlpolybrene was added. The retroviral supernatant was added daily,following removal of medium and washing of the cultured cells, for 1-7d. Control cultures were incubated with fresh medium. Two days afterthese infections were completed, equal numbers of H460a cells wereseeded into a selective medium containing 300 μg/ml G418 or nonselectivemedium for 10-14 d. The infection efficiency for an infected cellpopulation was measured by dividing the number of G418-resistantcolonies by the number of colonies growing in the absence of selection.

[0153] 4. PCR Analysis of Genomic DNA From Transduced H460a Cells

[0154] Genomic DNA was isolated by SDS-proteinase K lysis of H460a cellsfollowed by phenol-chloroform extraction. One microgram of genomic DNAwas placed in a total volume of 100 μl containing 50 Mm KCl, 10 MmTris-Hcl, 1.5 Mm MgCl₂, 0.1% gelatin, 20 Mm deoxyribonucleosidetriphosphates, 660 ng each of two neomycin phosphotransferase (neo-r)oligonucleotide primers (neo 1: CAAGATGGATTGCACGCAGG; neo 5:CCCGCTCAGAAGAACTCGTC), and 2.5 units of Taq DNA polymerase. The tubeswere cycled 35 times through 94° C. for 1 min, 50° C. for 1 min, and 72°C. for 2 min. The PCR products (15 μl) were electrophoresed on 2% gel(1% agarose, 1% nusieve GTG agarose) stained with ethidium bromide. TheDNA was transferred onto a nitrocellulose membrane and hybridized with³²P-nick translated neo gene probe (Hind III/Sma I neo gene fragment ofPsv2-neo plasmid DNA) in 6×SSC, 10×Denhart's solution, 0.1% SDS, 100μg/ml salmon sperm DNA, and 25 Mm NaH₂PO₄ at 65° C. for 3 h.

[0155] 5. Slot Blot Hybridization of Poly(A⁺) RNA

[0156] Poly(A⁺) RNA was isolated from the cell lines. The RNA wasdenatured with 50% formamide, 6% formaldehyde, and 1×SSC at 68° C. for15 min, then blotted onto nitrocellulose membranes (8 μg, 4 μg, or 2 μg)using a slot blot apparatus. The filters were prehybridized andhybridized at 64° C. for 8-12 h with a ³²P-end-labeled 42-bp K-ras exon2 DNA oligonucleotide probe in 1×SSPE, 2×Denhardt's solution, 1% nonfatdry milk, 10% dextran sulfate, 2% SDS, 200 μg/ml salmon sperm DNA, 200μg/ml yeast tRNA, and 200 μg/ml polyadenylic acid. They were then washedtwice in 1×SSPE, four times in 0.2×SSPE for 30 min at room temperature,and finally with 0.1×SSPE for 30 min to 1 h at 47-58° C. The filterswere exposed for 2-3 d at 80° C. A β-actin probe was used to reprobe thefilters to confirm equal loading of RNA.

[0157] 6. Immunoblot Analysis of ras Protein

[0158] Protein extracts were prepared by lysing cells in Laemmli buffer(130 Mm Tris-Hcl, Ph 6.8; 2% SDS; 10% glycerol). The extracts wereboiled for 5 min, cooled in ice and cleared by centrifugation at10,000×g for 15 min. The protein concentrations were calculated bybovine serum albumin protein assay. One hundred micrograms of proteinwas size-fractionated by 12.5% SDS-polyacrylamide gel and electroblottedonto nitrocellulose membranes, ras-specific p21 protein was detectedusing either a K-ras or a pan-ras-specific p21 monoclonal antibody(oncogene Science, Mahasset, N.Y.) followed by horseradishperoxidase-labeled goat anti-mouse second antibody (Pierce, Rockford,Ill.). The change in K-ras p21 levels was determined by measuringabsorbance with a video densitometer (Model 620, Bio-Rad, Richmond,Calif.).

[0159] 7. Proliferation and Soft Agarose Colony Formation by H460a cells

[0160] Parental and infected H460a cells (10³/well) which were selectedor not selected with 300 μg/ml G418 were seeded and grew in 12-wellplates for 7 d. Cells were harvested and counted at different days.Human fibroblast cell line MRC-9 was used as a control. Aliquots of5×10⁴ cells were mixed with 0.35% agarose in RPMI 1640 medium and platedover a base layer of 0.7% agarose and culture medium hardened in 60-mmdishes. Colonies (>50 cells) were counted using a phase contrastmicroscope 10-14 d later.

[0161] 8. Tumorigenicity of H460a cells in Orthotopic Lung Cancer Model

[0162] A model of orthotopic lung cancer growth in nu/nu mice was usedto measure tumorigenicity of these cells. Balb/c nu/nu mice wereirradiated with 350 Cgy of whole-body irradiation from a ⁶⁰Co source at127 cGy/min. After being anesthetized with methoxyflurane, theH460a-antisense-LNSX construct, the H460a cells infected by theretroviral vector alone, or H460a parental cells were injectedendotracheally (10⁵/mouse) using a 27-gauge blunt needle. Themediastinal block was harvested after 4 wk and tumor growth was measuredwith linear calipers in two orthogonal directions without knowledge ofthe animal treatment group.

[0163] B. Results

[0164] 1. Construction and Generation of β-actin K-ras Antisense LNSXReplication-defective Retrovirus

[0165] Recombinant plasmid clones were constructed by subcloning awild-type 1.8-Kb K-ras genomic DNA segment carrying second and thirdexons together with flanking intron sequences and a β-actin promoter inantisense orientation into an LNSX retrovirus vector in two orientations(FIG. 4). The plasmid DNA was analyzed by restriction enzyme mappingwith controls of LNSX plasmid DNA only and the β-actin K-ras antisenseApr-1-neo vector. β-actin K-ras antisense LNSX was constructed in twodifferent orientations, both of which included the 4-Kb β-actinpromotor, the 1.8-Kb K-ras fragment, and a 6-Kb LNSX vector fragment.The digested DNA was transferred to a nitrocellulose membrane andhybridized with a 1.8-Kb ³²P-nick-translated genomic K-ras probe.Orientation a has the K-ras 5′ end adjacent to the SV40 promoter of LNSXand thus is placed in a reverse orientation((LTR_neo_SV40_K-ras_β-actin_LTR). Orientation b has the β-actinpromoter adjacent to the SV40 promoter (LTR_neo_SV40_βactin_K-ras_LTR).

[0166] The amphotropic retrovirus was produced by transfection of theGP+envAm12 packaging cell line with this recombinant DNA. To increaserecombinant retrovirus production, amphotropic β-actin K-ras antisenseLNSX (orientation a) GP+envAm12 cells were co-cultivated with ecotropicΨ-2 for 1 month. This mixed-cell pool was selected by HXM medium withhygromycin B and G418. The highest viral titer generated by testing theselected colonies was 9×10⁶ CFU as determined by transduction andselection of NIH-3T3 cells.

[0167] Replication-competent virus produced by GP+envAm12 was measuredby infection of fresh NIH-3T3 cells with medium conditioned in NIH-3T3cell cultures infected by recombinant retrovirus and selected by G418for 10-14 d (short-term assay). In a more sensitive long-term assay,NIH-3T3 cells were infected with the medium conditioned byGP+envAm12-producing cells, after which they were passaged for 1 monthto allow for the spread and amplification of a rare recombinantwild-type virus in the culture. Medium collected from these NIH-3T3cells was used to infect fresh NIH-3T3 cells. Both the short-term andlong-term assays showed that no detectable replication-competentretrovirus was produced by GP+envAm12 cells.

[0168] 2. Infection Efficiency in H460a cells.

[0169] H460a cells were infected with recombinant LNSX-antisenseretrovirus by incubating with viral stocks in the presence of 8 μg/mlpolybrene. The infection efficiencies of H460a cells at varying virus toH460a cells ratios (V/T) of 1:10, 1:1, 10:1, and 100:1 were 7.5±1%,26±1.2%, 53±11%, and 57±13% after a single cycle of infection (FIG. 5A).The efficiency increased with higher V/T ratios and plateaued at the10:1 V/T ratio. The infection efficiency increased also with the numberof infection exposures at the same V/T ratio (100:1) (FIG. 5B).Infection efficiencies of 9735 15% and 25±2.6% were achieved after fivecycles of infection of H460a cells at 10:1 and 1:10 V/T ratios,respectively.

[0170] 3. Detection of Transduced neo gene by PCR in Infected H460acells

[0171] Genomic DNA was isolated and amplified by PCR with neo 1 and neo5 oligonucleotide-primers. A 790-bp segment of the neo gene was detectedin transduced H460a cells, but not in parental H460a cells (FIG. 6A).Southern hybridization with a ³²P-nick-translated neo gene probeconfirmed the identity of the neo gene band (FIG. 6B), indicating thatthe inserted retrovirus gene was successfully integrated into H460agenomic DNA.

[0172] 4. Expression of K-ras Antisense RNA and Specific Inhibition ofK-ras Protein p21 in H460a cells

[0173] Poly(A+) RNA was extracted from parental and infected H460acells. The expression of K-ras antisense RNA was detected by slot blothybridization with a 42 bp K-ras exon 2 oligonucleotide probe (FIG. 7).The level of expression of K-ras antisense RNA in H460a cells infectedby the orientation (a) retrovirus was higher than that of H460a cellsinfected by the orientation (b) retrovirus. Reprobing the filter withthe β-actin DNA probe showed that each sample was loaded equally.

[0174] The inventors next analyzed the p21 protein level in these H460acells by immunoblot analysis. A K-ras-specific p21 monoclonal antibodywas used to determine the level of K-ras protein in parental andinfected H460a cells. K-ras p21 protein synthesis was reduced by 90% inthe H460a cells expressing high levels of K-ras antisense RNA(orientation a retrovirus) and by 30% in the H460a cells infected withorientation b retrovirus, compared with those of parental H460a cellsand H460a cells infected only by the LNSX vector retrovirus (FIG. 8A).The total ras protein production in these cells was also examined, usinga pan-ras monoclonal antibody, to determine whether a reduced level ofK-ras protein reflected any change in H-ras and N-ras p21 proteinsynthesis. The western blot analysis revealed that overall ras proteinlevels in all infected cells were only slightly decreased from the levelin H460a parental cells (FIG. 8B).

[0175] 5. Suppression of H460a Cells' Growth in Vitro and ColonyFormation in Soft Agarose

[0176] H460a cells expressing K-ras antisense RNA continued to be viablein culture. However, growth of H460a cells expressing high levels ofK-ras antisense RNA (orientation a) was reduced compared with that ofH460a cells infected with LNSX vector only and parental H460a cells(FIG. 9A). Transduction of the human lung fibroblast cell line MRC-9,which has a wildtype K-ras gene, with LNSX and LNSX-AS-K-ras (a) did notaffect proliferation of that cell line (FIG. 9B). Previous studies haveshown that expression of the 1.8-Kb K-ras fragment in the senseorientation does not affect proliferation or tumorigenicity of H460acells (Mukhopadhyay et al., 1991).

[0177] The effect of K-ras antisense RNA expression on the growth ofsoft agarose colonies of H460a cell lines was determined. Colonyformation in soft agarose was dramatically decreased in H460a cellsexpressing K-ras antisense RNA (number of colonies, orientation a:135±26; orientation b: 320±37) as compared with parental H460a cell line(1096±434) and H460a cells infected only with retroviral vector LNSX(1048±322) (FIG. 10).

[0178] 6. Suppression of Tumorigenicity in an Orthotopic Lung Cancernu/nu Mouse Model

[0179] Intratracheal inoculation of H460a cells in irradiated nu/nu miceresulted in the growth of endobronchial tumors with mediastinalextension in >80% of the mice after 4 wk. Twelve of 14 mice inoculatedwith parental H460a cells and seven of nine mice inoculated with H460acells infected with the LNSX vector developed tumors (Table 2). Three ofseven mice inoculated with H460a cells transduced with theLNSX-AS-K-ras(b) had tumors. Cells expressing the highest level ofAS-K-ras with the greatest reduction in p21 expression had the lowestincidence of tumor formation. Only three of 17 mice receivingH460a-LNSX-AS-K-ras(a) cells had tumors and the volume of these tumorswas much less than tumors in the control groups. Statistical analysis(chi-square) shows that there is a statistically significant difference(p<0.005) in tumorigenicity between H460a-LNSX-AS-K-ras(a) and thecontrol groups. TABLE 2 Tumorigenicity of H460a in orthotopic nude micemodel Cells Mice with Meanvolume Cell lines injected Tumors (%) (mm³)¹H460a 10⁵ 12/14 (86) 39.9 ± 4.25 H460a-LNSX 10⁵ 7/9 (78) 12.5 ± 2.2 H460a-LNSX-AS-K-ras (a) 10⁵ 3/17² (18) 2.95 ± 1.25 H460a-LNSX-AS-K-ras(b) 10⁵ 3/7 (43) 1.74 ± 1.5 

[0180] B. Discussion

[0181] A retroviral vector-mediated gene transfer system was developedto introduce a partial K-ras genomic sequence into lung cancer cell lineH460a, which has the K-ras gene mutation at codon 61. The K-ras sequencecarries second and third exons together with flanking intron sequencesand a β-actin promoter in antisense orientation. The transduced K-rasantisense gene was integrated and efficiently expressed in H460a cells.For H460a cells expressing AS-K-ras, K-ras-specific p21 proteinexpression was reduced more than 90%, whereas the total ras proteinproduction decreased only slightly relative to the control group(parental H460a cells and those infected only by the retroviral vector).Specific inhibition of oncogene (e.g.,N-ras, H-ras) expression byantisense oligonucleotides has been reported by a few laboratories, butthe short biological half-life and low transfer efficiency ofoligonucleotides in the cell were problems in those studies(Saison-Behmoaras et al., 1991; Chang et al., 1991; Neckers et al.,1992).

[0182] In the presently disclosed retroviral gene transfer system, hightransfer efficiency, prolonged expression of K-ras antisense RNA, andinhibition of K-ras p21 protein were achieved, particularly through theuse of reverse orientation constructs. Cells expressing the antisenseK-ras construct have been grown in continuous culture for over 6 months.The expression of the neoplastic phenotype of the H460a cell line,including growth rate, ability to form colonies in soft agarose, andtumorigenicity in nude mice, were dramatically reduced. Previous studieshave shown that cancer cells often have multiple genetic alterations.Therapy directed toward oncogenes will be practical only if therapeuticeffects occur with targeting of one or two genes. In this case reversalof a single genetic lesion resulted in suppression of critical featuresof the malignant phenotype.

[0183] The inventors results indicate that the expression of the mutatedK-ras protein plays an important role in the oncogenesis and growth ofcell line H460a. When human fibroblast cell line MRC-9 and NSCLC cellline H322a, which has a wildtype K-ras gene, were infected by theLNSX-antisense retrovirus, the growth curves were not significantlydifferent from that of the control cells. Thus, this construct candistinguish among closely related members of the ras family. Continuedviability of cells expressing AS-K-ras suggests that other closelyrelated members of the ras family may subsume the function of K-ras.

[0184] It is unlikely that any therapy targeting oncogenes or theirproducts will be absolutely specific for cancer cells. If other genescan compensate for loss of normal function by a specific oncogenemediated by an antisense construct, the harmful effects of the therapywill be reduced. Additional support for this concept comes from a recentstudy by Soriano and coworkers (Soriano et al., 1991) in whichtransgenic mice were created that lacked a functional c-srcproto-oncogene. The resulting developmental defect in the mice wasosteopetrosis. The ubiquity of c-src, its high degree of conservationamong species, and its role in mitosis suggest that inactivation wouldbe lethal, but this was not the case; viable mice were recovered. Apossible explanation is that other closely related nonreceptor tyrosinekinases such as yes and fyn can compensate for loss of c-src.

[0185] Efficient transfer of constructs that can modify expression ofoncogenes and tumor suppressor genes is critical to the analysis of thefunctional role of these genes and the potential therapeutic use ofthese constructs. We found that infection efficiency could achieve 97%using a multiple infection protocol. After one exposure, efficiency ashigh as 57% was achieved at a 10:1 V/T ratio, with little additionalincrease in efficiency obtained by increasing the V/T ratio to 100:1,indicating that such factors as the quality of the virus preparation andthe proliferation status of the H460a cells may affect the infectionefficiency. In the low V/T ratio (1:10) assay, infection efficiency ofabout 25% was obtained after five exposures. Ratios of retrovirus totumor cells or premalignant cells such as these are achievable withregional therapy in the setting of minimal residual cancer orpremalignant conditions. This revealed that, in the clinical setting,even if the high V/T ratio cannot be achieved, a satisfactory infectionefficiency may be obtained by multiple infection exposures. Not all thepatient's cancer cells will be in the proliferative stage at eachinfection exposure, and the retrovirus may selectively infect only theproliferating cells (Miller et al., 1990). Multiple exposures to theretrovirus can address this problem and maximize the number oftransduced tumor cells. The use of a promoter which is commonlyexpressed in epithelial cells may also contribute to efficientexpression in human cancer cells of epithelial origin.

[0186] The high titer (9×10⁶ CFU/ml) of the producer cell line wasobtained by “ping-pong” infection between the amphotropic packaging cellline GP+envAm12 and ecotropic packaging cell line Ψ-2. The titer was 100times more than those of cell lines produced by GP+envAm12 before“ping-pong” infection. A similar result was reported by Bodine et al.,but in their assay all of the high-titer cell lines after ping-ponginfection also produced replication-competent viruses (Bodine et al.,1990). In the present system, no detectable replication-competent viruswere produced even in the stringent long-term assay. This may be due tothe safety of GP+envAm12, in which the Moloney murine leukemia virusgag, pol gene, and 4070A amphotropic env gene are separated on Pgag-polgpt and PenvAm, two different plasmids, and the packaging signals and3′long-terminal repeats are removed. The three specific recombinationevents required to generate replication-competent viruses are unlikelyto occur in this system (Markowitz et al., 1988).

[0187] A very interesting finding is that, of the two orientations ofthe construct in the recombinant retroviral vectors[LTR_neo_SV40_-ras_β-actin_LTR (a) and LTR_neo_SV40_β-actin_K-ras_LTR(b)], the orientation (a) vector showed higher transfection efficiency,higher virus titer, and higher K-ras antisense RNA expressionefficiency. It is possible that the SV40 promoter may suppress theβ-actin promoter as described in other systems (Emerman et al., 1984).However, the SV40 promoter is not as active as the β-actin promoter, andtherefore this effect may have some degree of promoter specificity(Gunning et al., 1987; Emerman et al., 1986). If some sense transcriptswere produced by this promoter in orientation (a), the splicing of theintron sequence would render the transcripts unable to hybridize withthe antisense transcripts. The effectiveness in the reduction of K-rasp21 protein by orientation (a) supports the absence of this type ofinhibitory effect. Interestingly, the use of a β-actin promoter inorientation (b) with an LNL6 retrovirus yielded low rates of infectivityand low levels of gene expression (Owens et al., 1991).

[0188] According to the original “seed and soil” hypothesis proposed byPaget in 1889, organ-site specific implantation of tumor cells isessential for optimal growth and progression of tumors in vivo (Paget,1989). This concept has been widely supported by numerous studies inmetastatic tumor models (Fidler, 1986) and, recently, athymic nude micemodels have been used to study the orthotopic propagation of selectedhuman solid tumors, including lung cancer (Howard et al., 1991). Wesuccessfully used an intratracheal model for the orthotopic propagationof human lung cancer H460a cells in irradiated nude mice to assess thetumorigenicity of the transduced cells. The H460a cells grew well in themodel, and the tumorigenicity of H460a cells expressing K-ras antisenseRNA was dramatically decreased. Further studies using retroviral vectorsas a regional delivery method for K-ras antisense gene expression invivo are in progress in our laboratory. The orthotopic in vivo model inuse closely resembles the clinical setting, allowing a furtherassessment of the feasibility of using the recombinant retrovirustherapeutically in lung cancer.

EXAMPLE III Clinical Protocol for Modification of oncogene and TumorSuppressor Gene Expression in Non-Small Cell Lung Cancer

[0189] This example is provided to demonstrate a protocol foradministering and assessing the efficacy and toxicity of theintralesional administration of retroviral constructs containingantisense (AS) K-ras (for tumors with mutated K-ras) and wildtype p53(wtp53) (for tumors with mutated or deleted p53) into residualendobronchial NSCLC which obstructs a bronchus and which is refractoryto conventional therapy.

[0190] A. Downregulation of Activated K-ras/expression With an AntisenseConstruct

[0191] 1. Gene construct

[0192] The retroviral vector construct contains the AS-K-ras fragmentwith its β-actin promoter inserted into the LNSX vector (Miller et al.,1989; Palmer et al., 1987). The orientation of the insert is such thatthe transcription of the AS-K-ras is driven by the β-actin promoter inan orientation that is reverse with respect to transcription from theretroviral LTR.

[0193] 2. Packaging

[0194] Because recombination events may lead to the production of areplication-competent virus, a safe and efficient amphotropic packagingcell line is necessary for transfer of exogenous genes into human cancercells. The packaging cell line employed is constructed so the gag-poland env genes are separated on two different plasmids (Markowitz et al.,1988). The packaging signals and 3′ LTRs have also been removed; thiswas done by transfection of NIH 3T3 cells by a plasmid containingMoloney murine leukemia virus gag and pol genes and a separate plasmidcontaining the env gene. The GP+envAM12 clone that produces high levelsof env protein was selected to be used as the packaging cell line. Thecombination of mutations for the two plasmids requires at least threerecombination events between the helper plasmids and the retroviralvector; the improbability of this sequence of events essentiallyeliminates the possibility of replication-competent virus production.The presence of functioning retroviral genes in the packaging cell linewill be monitored by an assay for reverse transcriptase production andby immunoprecipitation of env protein by metabolic labeling andimmunoprecipitation with anti-env antiserum (Markowitz et al., 1988).

[0195] Continued absence of infectious virus will be determined fromtransfection-infection experiments. A neo-containing vector will betransfected into GP+envAM12 cells; colonies will be selected with G418.The supernatants will be used to infect NIH 3T3 cells. Selection withG418 will be done after one month to ensure the survival of rarerecombinants that do not have the neo gene but subsequently infectneo-positive cells. Supernatants from the infected NIH 3T3 cells shouldnot be infectious. These secondary supernatants will be used to infectnaive NIH 3T3 cells. Lack of infectivity will indicate absence ofreplication competent virus.

[0196] A protocol for generating retroviral particles is as follows:

[0197] (1) GP+envAm12 cells are grown in Dulbecco's modified Eagle'smedium containing 10% newborn calf serum, 15 μg/ml hypoxanthine, 250μg/ml xanthine, and 25 μg/ml mycophenolic acid and selected in 200 μg/mlhygromycin.

[0198] (2) Vectors are transfected by electroporation.

[0199] (3) G418 (400 μg/ml) selection is begun 48 hr after transfectionand colonies are expanded 10 to 14 days later.

[0200] (4) The viral titer is tested by infecting NIH 3T3 cells. Afterproducer cells are semiconfluent, medium is replaced with Dulbecco'smodified Eagle's medium containing 10% newborn calf serum but withoutG418. Cells are seeded at 5×10⁵ in 60-mm dishes. The medium is removed18 hr later, filtered (0.45 micron), and diluted serially (10² to 10⁷).One milliliter of medium is applied to cells. Polybrene (8 μg/ml) isadded. Cells are incubated for 2 hr at 37°, and then 4 ml of freshmedium is added.

[0201] (5) After 48 hr cells are replated onto 100 mm tissue culturedishes and selected with G418. Previous human studies have used thePA317 producer cell line. This cell line is preferred because of theextensive experience with its use and prior approval for human use.

[0202] 4. Preclinical studies

[0203] The 2 Kb K-ras fragment (genomic exons 2 and 3) with a β-actinpromoter was cloned into the LNSX retroviral vectors in bothorientations. The p53 cDNA with its β-actin promoter was cloned into theLNSX retroviral vectors in both orientations. Both the LNSX-AS-K-ras andthe N2A-AS-K-ras have been successfully packaged in the GP+envAm12packaging cell line. Initial titers ranged up to 10⁴. By using a“ping-pong” technique, the titer of the LNSX-AS-K-ras supernatant wasincreased to 5×10⁶. In this technique, supernatants from the GP+envAm12packaging cell line were used to transduce the ecotropic packaging cellline ψ2 (Mann et al., 1983). Supernatants from this transduction wereused again to transduce GP+envAm12. Both constructs were then transducedinto H460a cells. Specific expression of K-ras AS RNA was shown by slotblot analysis using vector only negative controls and a β-actin probefor a loading control. Western blotting studies showed that expressionof the K-ras p21 protein was specifically reduced. Next the effect ofmultiple cycles of transduction on transduction efficiency was assessed.Transduction efficiency was assessed on a functional level (FIG. 11).H460a cells were transduced with either LNSX or LNSX-AS-K-ras daily for4 consecutive days. Cells grew for 7 days without selection.

[0204] The percent reduction in the growth fraction of the AS transducedcells reflects the efficiency of transduction as growth of a selectedpopulation of AS transduced cells does not occur during this timeperiod. The growth of the unselected AS transduced cells was less than20% at 7 days. Thus, the simple manipulation of exposing cells to thepackaged retrovirus for 4 consecutive days caused a striking increase intransduction efficiency. In a subsequent experiment H460a cells weretransduced daily for 7 consecutive days with LNSX-AS-K-ras and thenselected for colony formation in G418 (FIG. 12). Colonies were comparedto H460a cells that were not exposed to selective medium. Followingselection the efficiency of colony formation by the transduced cells was98%. This reinfection strategy is applicable to regional therapy. Theapparent low toxicity of the retroviral constructs should permitmultiple treatments. It is anticipated that the residual number ofendobronchial tumor cells can be reduced to <10⁷ so that an excess ratioof retroviral particles to proliferating tumor cells can be achieved.

[0205] The tumorigenicity of the transduced H460a cells was studied inan orthotopic lung cancer model. Intratracheal inoculation of H460acells in irradiated (350cGy) nu/nu mice resulted in the growth ofendobronchial tumors with mediastinal extension in >80% of mice after 4weeks. The H460a-AS-LNSX, H460a-LNSX, and H460a cells (10⁵/mouse) wereinjected endotracheally and the mediastinal block was harvested after 4weeks. Mice were assessed for tumor growth without knowledge of thetreatment group. Seven of 9 mice inoculated with H460a-LNSX (mean volume12.5±2.2 SE mm³) and 12 of 14 mice inoculated with H460a parental cells(mean volume 39.9±4.25 SE mm³) had tumors. Only 3 of 17 mice receivingH460a-AS-LNSX cells had tumors (mean volume 2.95±1.25 mm³). From thesestudies, it is concluded that 1) retroviral gene transduction can beused to express anti-sense constructs in human tumor cells at levelsthat mediate a biologic effect; 2) AS-mediated inhibition of activatedK-ras expression effectively inhibits proliferation and tumorigenicityof human cancer cells. Expression of the AS-LNSX expression in the H460acells has been stable up to 6 months.

[0206] B. Restoration of Expression of wtp53 Gene Product

[0207] 1. Preliminary studies with plasmid DNA

[0208] The p53 gene is the most commonly altered gene yet described inhuman cancers. To study this gene, a cell culture model system of celllines varying in p53 expression was established. The H322a lungadenocarcinoma cell line expresses the mutant p53 protein as shown bythe presence of high levels of endogenous p53 mRNA and phosphorylatedprotein. We showed that the H322a cell line has a G:T transversion atcodon 248 (Arg to Leu) with absence of the wildtype allele. The H358acell line has a homozygous p53 deletion. The H460a and H226b cell linesare homozygous for the wildtype p53. Expression vectors for sense(S-p53) and antisense p53 (AS-p53) cDNA with a β-actin promoter wereconstructed to study the effect of wtp53 expressed in lung cancer cellswith mutant or deleted p53 and the effects of reducing wildtype andmutant p53 expression.(Mukhopadhyay et al., 1991)

[0209] Stable transfectants of p53 mutant cells (H322a) or deleted p53(H358) expressing S-p53 could not be rescued. Failure to isolatecolonies expressing sense p53 RNA in cells with homozygous mutant ordeleted alleles shows that wtp53 can suppress transformation in cancercells expressing a mutant p53 or having a homozygous p53 deletion.

[0210] In general, transfection with AS-p53 reduced colony formation(10-fold) by cells with endogenous mutant p53. This indicates thatexpression of mutant p53 contributes to the transformed phenotype. Asexpected, cells with wtp53 (H226b) showed increased tumorigenicity whentransfected with AS-p53. The H226b cells expressing AS-p53 growsignificantly more rapidly in nu/nu mice than the cells transfected withthe control plasmid. This indicates that elimination of the wtp53 geneproduct enhances features of the malignant phenotype.

[0211] The inventors studies showed that wtp53 is dominant and cansuppress the malignant phenotype in cells with mutant or deleted p53.The presence of the mutant p53 confers transforming potential to thegene product, which can be suppressed by AS-p53. Thus in cancer cellsboth the absence of wtp53 and the presence of certain p53 mutations mayenhance the malignant phenotype.

[0212] 2. Gene construct

[0213] The retroviral vector construct contains p53 cDNA with itsβ-actin promoter inserted into the LNSX vector (Miller et al., 1989;Palmer et al., 1987) in a reverse orientation, in essentially the samemanner as described for the p21 AS embodiments.

[0214] 3. Packaging

[0215] See section A.2. above

[0216] 4. Preclinical studies

[0217] The LNSX-p53 and the DC-p53 were transduced into H322a (mutantp53), H358a (deleted p53), and H460a (wt p53). H322a cells thatunderwent one cycle of infection with the wtp53 construct but withoutG418 selection had an over 3-fold reduction in proliferation compared tocells that received either the unmodified vector or no treatment. Twocycles of transduction without G418 selection resulted in a 5-foldreduction in proliferation (FIG. 13). A similar result was observed forthe H358a cell line when transduced with LNSX-p53. The proliferation ofthe H460a cell line which has a wildtype p53 was not altered bytransduction with any of the p53 retroviral constructs (FIG. 14). Thus,retroviral mediated gene transfer of wtp53 into human lung cancer cellswith deleted or mutated p53 significantly reduces the proliferation ofthose cells. The expression of the mutated p53 protein is uniform incultured cell lines as detected by immunohistochemistry. In fresh lungtumors that express high levels of p53 protein, expression is detectedin >90% of cells.

[0218] A critical question is the ability of the retroviral constructsto transduce established tumor cells in vivo. This question wasaddressed by injecting H460a (10⁵) cells in the mouse right mainstembronchus followed 3 days later by lavage with LNSX retroviralsupernatant (10⁶ CFU in 0.1 ml). LNSX was used so that the neo genecould be used as a marker for transduction. It was necessary to recovertumor cells for analysis so that the AS construct was not used. Tumorswere harvested and the presence of the neo gene was assessed by Southernhybridization. The neo gene was detected in the DNA from the H460a cellsindicating successful transduction of the retrovirus 30 days afterlavage. Although this data is encouraging, the model has limitations.Direct injection of endobronchial tumor is not possible in this model.Other sites of direct injection do not accurately simulate the milieu ofendobronchial lung cancer. Thus, definitive answers concerning efficacymust be obtained through this clinical trial.

[0219] C. Treatment Plans

[0220] In proposed preferred treatment protocols, patients will undergobronchoscopy to assess the degree of obstruction. As much gross tumor aspossible should be resected endoscopically. Patients should preferablyundergo bronchoscopy under topical or general anesthesia. A Stifcor™transbronchial aspiration needle (21 g) will be passed through thebiopsy channel of the bronchoscope. The residual tumor site will beinjected with 10⁷ CFU of the appropriate retroviral supernatant. Thevolume will be no greater than 10 ml. Protamine will be added at aconcentration of 5 μg/ml. This is 0.2% of the amount given intravenouslyto reverse heparinization.

[0221] Injections will be circumferential and will be intratumor andsubmucosal. The AS-K-ras supernatant will be used for K-ras mutationsand the p53 supernatant will be used for p53 mutations. The injectionswill be repeated daily for five consecutive days. The treatment will berepeated monthly.

[0222] 1. Criteria for response and toxicity

[0223] There are various criteria that one should consider as presentingthe existence of a need for response or the existence of toxicity. Toassist in determining the existence of toxicity, the tumor bed should bephotographed prior to a course of therapy. The longest diameter and itsperpendicular will be measured. Size will be reported as the product ofthe diameters. From these date, one can calculate from these numbers therate of regrowth of the tumor.

[0224] The time to progression can also be measured from the firstobservation with reduction in tumor bulk until there is evidence ofprogressive disease. Progressive Disease is defined as an increase of≧25% in the sum of the products of the diameters of the measured lesion.Patients must have received at least two courses of therapy before adesignation of progression is made. The survival of patients will bemeasured from entry into protocol.

[0225] 2. Potential risks of retroviral gene transduction

[0226] The possibility of causing malignancy in normal cells secondaryto random insertion of the retroviral vector in the genome existsalthough this risk is thought to be very low. Tests of viral supernatantwill be conducted to assure that no replication competent virus ispresent. Non-replicating bronchial epithelial cells will not take up thevector.

[0227] 3. Risk from murine retrovirus.

[0228] The retrovirus derived from the Moloney murine leukemia virus ismodified so that it no longer contains intact viral genes. Thus, itcannot produce an intact infectious virus. Assays may be performed onthe retroviral vector supernatant and the packaging cell to insure thatreplication competent virus is not present. Extensive safety studieshave been performed on this retroviral construct in primates. Largeinfusions of infectious murine amphotrophic virus produce no acutepathologic effects. Primates have also received retroviral gene-modifiedautologous bone marrow cells with no evidence of toxicity as long as 4years after infusion.

[0229] 4. Efficacy of aminoglycoside antibiotics.

[0230] The neomycin resistance gene product, neomycinphosphotransferase, phosphorylates the 3′ hydroxyl group of theaminohexose I of neomycin and its analogues. Amikacin, but notgentamicin and tobramycin which do not contain an hydroxyl at the 3″position, is inactivated. Thus, induction of the neomycin resistancegene would not exclude aminoglycosides or any other conventionalantibiotic from use in these patients.

[0231] 5. Criteria for discontinuing therapy

[0232] There are various criteria that one should consider employing inmaking a decision to discontinue therapy. For example, an increase inthe endobronchial tumor after a minimum of 2 or more courses of therapy,or the development of unacceptable toxicity defined as unpredictable,irreversible, or Grade 4. Patient refusal of therapy due to a specifictoxicity should be graded as 4 and an explanatory note recorded. Oneshould also consider discontinuing therapy upon the occurrence ofsignificant hemoptysis, coagulopathy, or progressive postobstructivepneumonia

[0233] The present invention has been disclosed in terms of preferredmodes found to work well in the practice of the invention. However,numerous modifications and changes in the steps, procedures used andmaterial will become apparent to those of skill in the art in light ofthe disclosure. All such modifications are intended to be within thespirit of the present invention and scope of the appended claims.

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What is claimed is:
 1. A retroviral expression vector comprising a geneexpression unit which includes a selected gene under the control of aβ-actin promoter, the gene expression unit being positioned to effecttranscription of the selected gene in an orientation opposite that ofretroviral transcription.
 2. The vector of claim 1, wherein the selectedgene encodes an RNA molecule that alters the expression of a cellulargene.
 3. The vector of claim 2, wherein the selected gene encodes anantisense RNA molecule.
 4. The vector of claim 3, wherein the geneencodes an antisense RNA molecule that is complementary to a selectedcellular gene.
 5. The vector of claim 3, wherein the antisense moleculeis complementary to an oncogene sequence.
 6. The vector of claim 3,wherein the encoded antisense RNA molecule capable of selectivelyinhibiting the expression of selected gene product, the encodedantisense RNA molecule including a region that is complementary to andcapable of hybridizing with an intron region of the selected gene. 7.The vector of claim 6, wherein the selected gene product is a product ofa gene family member and encodes an intron region that is distinct fromintron regions of another family member, the antisense RNA moleculebeing capable of selectively inhibiting the expression of the selectedgene product over that of another member of the family.
 8. The vector ofclaim 6, wherein the encoded RNA molecule includes a sequence that iscomplementary to an entire intron.
 9. The vector of claim 8, wherein theencoded RNA molecule comprises a sequence that is complementary to exonregion sequences of the selected gene.
 10. The vector of claim 9,wherein sequences complementary to the intron and exon regions of theselected gene are adjacent, and includes a sequence that iscomplementary to an intron/exon junction of the selected gene.
 11. Thevector of claim 7, wherein the gene family comprises the ras, myc, erbor jun family of oncogenes.
 12. The vector of claim 5, wherein theoncogene is one which is activated by a point mutation.
 13. The vectorof claim 11, wherein the oncogene is a ras oncogene.
 14. The vector ofclaim 13, wherein the RNA encodes a sequence that is complementary to anintron region of the p21 K-ras oncogene that is not found in an intronof H-ras or N-ras.
 15. The vector of claim 14, wherein the intron regioncomprises a region from intron II of the p21 K-ras oncogene.
 16. Thevector of claim 15, wherein the molecule encodes sequences complementaryto exons II and III and intron II of K-ras.
 17. The vector of claim 1,wherein the selected gene encodes a recombinant protein.
 18. The vectorof claim 17, wherein the recombinant protein confers a selected trait.19. The vector of claim 18, wherein the selected gene encodesrecombinant wild-type p53.
 20. The vector of claim 1, wherein the geneexpression unit is positioned in an orientation that is opposite that ofretroviral LTR.
 21. The vector of claim 1, further defined as a vectorderived from Moloney murine leukemia virus.
 22. The vector of claim 1,further comprising a second gene expression unit which includes a secondgene, expressed from a retroviral long-term repeat.
 23. The vector ofclaim 22, wherein the second gene comprises a selectable marker gene.24. The vector of claim 23, wherein the selectable marker gene comprisesa neo gene.
 25. A pharmaceutical composition comprising the vector ofany one of claims 1-24, in a pharmacologically acceptable state.
 26. Amethod for the preparation of a retroviral expression vector comprisingconstructing a gene expression unit which includes a selected geneplaced under the control of a β-actin promoter, and positioning the geneexpression unit into a selected retroviral vector in an orientationopposite that of retroviral transcription.
 27. A method for theexpression a gene encoding a selected RNA, the method comprisingpreparing a retroviral expression vector that includes a gene expressionunit comprised of a selected gene under the control of a β-actinpromoter, the gene expression unit being positioned to effecttranscription of the selected gene in an orientation opposite that ofretroviral transcription, and expressing the selected gene.
 28. Themethod of claim 27, wherein the retroviral expression vector isexpressed through introduction into a host cell.
 29. The method of claim28, wherein cells into which the retroviral expression vector have beenintroduced are introduced into a host organism.
 30. The method of claim28, wherein the retroviral expression vector is introduced into a hostorganism.